circKLF9 m for evaluating fat deposition ability in bovine 6 Molecular probes for a modification site, kits and uses thereof

Abstract

The application relates to a kind of circKLF9 m 6 The molecular probe of A modification site, kit and its application are aimed at solving the technical problems of insufficient specificity, weak anti-linear RNA interference ability and insufficient detection accuracy in the prior art detection of bovine circRNA m 6 The application is aimed at m 6 The specific primer and complementary probe of the core region of A modification are designed, and the m 6 The target fragment of A modification site is specific and has high amplification efficiency.The application can quantitatively detect the m 6 The level of A modification and gene expression level, for circKLF9 m 6 The application provides a core technical means for the mechanism research of A modification regulating bovine fat production, and can also be applied to the evaluation of bovine fat deposition capacity and molecular marker assisted selection of high-quality beef cattle.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to a circKLF9m assay for assessing bovine fat deposition capacity. 6 Molecular probes, kits, and applications for A-modified sites. Background Technology

[0002] Adipose tissue in beef cattle is an important energy storage and endocrine organ, and a key indicator determining meat quality and economic value. Intramuscular fat (IMF), deposited between skeletal muscle fiber bundles, is a core indicator determining beef marbling, tenderness, juiciness, and flavor, and a key regulatory target for breeding high-end beef cattle. IMF deposition is essentially a dynamic process of preadipocyte proliferation and differentiation and lipid droplet maturation; accurately assessing its differentiation capacity and deposition level is a core aspect of beef cattle breeding.

[0003] Recent studies have shown that circular RNAs (circRNAs), a class of non-coding RNAs formed by the backsplicing of precursor RNA, possess a highly stable covalently closed circular structure and can exert regulatory functions by acting as miRNA sponges, regulating parental gene transcription, or interacting with RNA-binding proteins. Meanwhile, N6-methyladenosine (m... 6 A) is the most abundant and conserved epigenetic modification in eukaryotic RNA, widely involved in RNA splicing, nucleoplasmic transport, stability, translation, and degradation. circRNA's m 6 Dynamic modification has been proven to be an important mechanism for regulating lipid synthesis in ruminants.

[0004] Currently, RNA m 6 Methods for detecting A methylation include antibody-enriched immunoprecipitation methods (such as MeRIP-seq, m...). 6 Antibody-independent chemical or enzymatic detection methods (such as SELECT, MAZTER-seq, etc.) and antibody-independent methods (such as SELECT, MAZTER-seq, etc.). For specific RNA molecules, m... 6 Verification of A modification levels often employs MeRIP-qPCR technology: first, using m... 6 Antibody A was used to enrich fragmented RNA through immunoprecipitation, and then quantitative qPCR was used to detect the enrichment fold of the target RNA in the immunoprecipitation fraction and the input fraction, thereby determining its m 6 A semi-quantitative assessment of the modification level.

[0005] However, directly applying existing MeRIP-qPCR technology to bovine circRNA m 6When detecting A-modification, the following shortcomings still exist: ① Linear RNA interference is difficult to eliminate: Conventional MeRIP-qPCR cannot effectively distinguish circRNA from its homologous linear RNA. circRNA shares most of its sequence with its parental linear RNA. When using conventional primers for detection, residual linear RNA signals will severely interfere with circRNA m 6 A. Inaccurate determination of modification levels leads to false positive results; ② Lack of beef cattle-specific detection tools: Existing detection primers are mostly designed for human or mouse models, lacking high-confidence m-type primers for beef cattle-specific circRNAs (such as circKLF9). 6 A. Specific primers and probes for modified sites lead to insufficient sensitivity and specificity in bovine samples; ③ The detection process is not adapted to bovine tissue characteristics: Existing method parameters are mostly based on human or mouse tissues and have not been optimized for the characteristics of bovine skeletal muscle / fat tissue, which is rich in connective tissue and RNases, resulting in poor batch-to-batch reproducibility and low RNA enrichment efficiency; ④ It cannot be directly linked to breeding applications: There is currently no method to directly link bovine specific circRNA m 6 A standardized evaluation process for quantitatively linking modification levels with fat deposition traits makes it difficult to directly convert test data into molecular breeding indicators that can be used for early screening of beef cattle.

[0006] Therefore, it is necessary to develop circRNA m that is highly specific, sensitive, and suitable for bovine tissue samples. 6 A testing tool and standardized procedures are of great significance for promoting the advancement of molecular breeding technology for beef cattle.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] For the detection of bovine circRNA m using existing technologies 6 To address the technical problems of insufficient specificity, weak resistance to linear RNA interference, and insufficient detection accuracy during A-modification, the inventors discovered that a large number of circRNAs undergoing adipogenic differentiation... 6 The modification level A showed a significant temporal change, with a difference of m. 6 A-modified circRNAs were significantly enriched in core pathways such as adipocyte differentiation and lipid metabolism, confirming the m-modification of circRNAs. 6 Dynamic modification is an important regulatory mechanism in the lipid synthesis process of ruminants. 6A-methylation modification can not only affect the half-life of circRNA by regulating its stability, but also precisely regulate the biological function of circRNA in a variety of ways, such as changing its subcellular localization, enhancing the translational activity of IRES elements, and regulating its interaction with RNA-binding proteins, thereby affecting the proliferation, differentiation, and lipid droplet formation of bovine adipocytes.

[0009] To this end, the inventors systematically identified two conserved m on the mature transcript of bovine circKLF9 using SRAMP bioinformatics prediction combined with MeRIP-seq sequencing technology. 6 A modification site; based on its sequence characteristics, an effective amplification method for circKLF9 m is designed. 6 A specific primer for the modified site; melting curves showed no nonspecific amplification and primer dimers, indicating high primer specificity and amplification efficiency; this primer and detection method can effectively distinguish circKLF9 m in bovine longissimus dorsi muscle tissue with high and low fat content. 6 The modification level and gene expression level of A revealed the m of circKLF9 6 A modification is closely related to its expression regulation and lipid deposition.

[0010] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: Provides a circKLF9 m for evaluating bovine fat deposition capacity 6 A molecular probe with a modified site, which is an oligonucleotide probe, whose nucleotide sequence is identical to that of bovine circKLF9 containing adenine at position 167 and / or 181. 6 The target sequence of the modified site is complementary to that of the target sequence, and the target sequence contains the adapter site of the circKLF9.

[0011] This invention also provides a circKLF9 m method for evaluating bovine fat deposition capacity. 6 A molecular probe with a modified site, which is a primer pair consisting of a forward primer F and a reverse primer R, and its nucleotide sequence is shown below: F: 5'-CGCTTCATGAGGAGCGACC-3', R: 5'-TTGATCATGCTGGGGTGGAA-3'.

[0012] The present invention also provides a primer-probe combination comprising the oligonucleotide probe and the primer pair.

[0013] The present invention also provides a kit comprising the oligonucleotide probe, the primer pair or the primer-probe combination, as well as RNase R enzyme and anti-m 6At least one of the following: antibody A, RNA fragmentation reagent, reverse transcription reagent, and real-time quantitative PCR premix.

[0014] This invention also provides a bovine circular RNA circKLF9 m 6 The method for detecting A methylation levels includes the following steps: (1) Extract total RNA from the bovine samples to be tested; (2) Remove linear RNA from the total RNA of the bovine samples to be tested and enrich circular RNA; (3) The sample enriched with circular RNA is processed into fragmented RNA samples; (4) Use anti-m 6 Antibody A was used to perform immunoprecipitation on the fragmented RNA sample to obtain immunoprecipitated RNA; (5) The immunoprecipitated RNA was purified and reverse transcribed to obtain cDNA; (6) The cDNA is amplified by real-time quantitative PCR using the molecular probe or the primer-probe combination described above; (7) Calculate the m of circKLF9 based on the real-time quantitative PCR amplification results. 6 A. Methylation modification level.

[0015] Furthermore, the bovine sample to be tested is the longissimus dorsi muscle tissue of a bovine.

[0016] Further, the reaction system for real-time quantitative PCR amplification in step (6) includes: 1 μL each of forward and reverse primers with a concentration of 10 μM, 10 μL of 2×SYBR Premix Ex Taq II or PCR premix containing fluorescently labeled probes, 2 μL of cDNA template and 6 μL of sterile water.

[0017] Further, the reaction procedure for real-time quantitative PCR amplification in step (6) is as follows: pre-denaturation at 95℃ for 5 min; followed by 40 cycles, each cycle including denaturation at 95℃ for 10 s and annealing extension at 68℃ for 20 s; if oligonucleotide probe method is used, fluorescence signal is collected during the annealing extension stage.

[0018] The present invention also provides the use of the molecular probe, the primer-probe combination, or the kit in the preparation of products for detecting and evaluating bovine fat deposition capacity or intramuscular fat content.

[0019] Furthermore, the product is used to detect the m of circKLF9 in the sample to be tested. 6 A modification level and / or expression level were used to assess intramuscular fat content in cattle; among which, the m of circKLF9 was... 6The level of A modification was positively correlated with the fat deposition capacity of cattle, and / or, the expression level of circKLF9 was negatively correlated with the fat deposition capacity of cattle.

[0020] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Target-specific and primers / probes highly specific and efficient: This application uses MeRIP-seq combined with bioinformatics analysis to accurately identify a high-confidence m-target on bovine circKLF9. 6 The A-modification site (adenine at position 167 and / or 181) was identified, clearly defining the corresponding adenosine nucleotide position and its DRACH motif characteristics, providing a reliable molecular target for the detection system. Based on this, primer pairs and complementary probes strictly spanning the circKLF9 linker site were designed. Experimental verification showed that this design effectively avoids interference from linear homologous sequences, exhibits high amplification efficiency, a single peak in the melting curve, no obvious primer dimers or non-specific amplification, and significantly improves the specificity of the detection.

[0021] 2. Dual-strategy synergy, high specificity and ability to distinguish between high and low fat: This approach differentiates circRNA from its homologous linear RNA mRNA using existing MeRIP-qPCR technology. 6 To address the issue of weak IMF signaling ability, this application employs a dual strategy of RNase R restriction pretreatment combined with a reverse primer at the crosslinker site. This ensures that the detection signal originates solely from the circular RNA transcript, avoiding false-positive interference caused by residual linear RNA at the source. Experiments show that this method can effectively distinguish between differences in lipid deposition between individuals with high IMF and those with low IMF. p <0.01), the detection specificity and discrimination ability are significantly better than existing general methods.

[0022] 3. High sensitivity and good repeatability, with a process suitable for standardization and promotion: Addressing the characteristics of low circRNA abundance in bovine skeletal muscle / adipose tissue samples and the presence of interfering factors such as connective tissue and RNases, this application clarifies primer design parameters, qPCR reaction system, and amplification conditions, achieving low-abundance circRNA... 6 Precise quantitative detection of RNA modification levels. Simultaneously, key steps such as RNA extraction, fragmentation, immunoprecipitation, magnetic bead blocking, and RNA purification have been systematically optimized, effectively reducing intra- and inter-batch variability. Furthermore, core reagents have been pre-formulated and integrated into a standardized kit, making it easy to use and suitable for widespread application in molecular biology laboratories and breeding testing institutions.

[0023] 4. Revealing regulatory relationships and directly empowering molecular breeding assessment in beef cattle: This application further discovered that the target circKLF9 m 6The level of A modification and its expression level are correlated with the fat deposition capacity of bovine muscle tissue, revealing that "m 6 The regulatory axis connecting "A modification—circKLF9 expression—lipid deposition" provides new insights into the epitranscriptional regulatory mechanism of intramuscular fat formation in beef cattle. Based on the above correlation, the primers, kits, and detection methods provided in this application can be directly applied to assess the fat deposition capacity of beef cattle germplasm resources, early screening of superior individuals, and prediction of breeding value. This provides a novel epigenetic marker for marker-assisted selection in beef cattle, demonstrating clear industrial application prospects and commercial value. Attached Figure Description

[0024] Figure 1 In one embodiment of this application, the SRAMP software predicts the circKLF9 m of cattle. 6 A is the result diagram of the modified site; where A is the predicted score distribution curve and B is the site information table.

[0025] Figure 2 This is a 1% agarose gel electrophoresis image of total RNA from bovine longissimus dorsi muscle tissue in one embodiment of this application; where M is DNA Marker, 1 is 28S RNA, and 2 is 18S RNA.

[0026] Figure 3 This is the qPCR melting curve of the circKLF9 specific primer in one embodiment of this application.

[0027] Figure 4 For example, in one embodiment of this application, the m of circKLF9 in the longissimus dorsi muscle tissue of cattle. 6 A. Quantitative results of enrichment level; where Low fat represents the low fat content group and High fat represents the high fat content group.

[0028] Figure 5 This is a quantitative result of the relative expression level of circKLF9 in the longissimus dorsi muscle tissue of cattle in one embodiment of this application; wherein, Low fat is the low fat content group and High fat is the high fat content group. Detailed Implementation

[0029] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0030] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and materials involved are all commercially available conventional products; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0031] The circKLF9 sequence used in this study is a full-length 652 bp circular RNA sequence derived from a single exon region of the Bos taurusKLF9 gene, containing four discrete 3'UTR regions. MeRIP-seq omics analysis showed methylation signals at this site in chr8:46587127–46587482.

[0032] Example 1: Bovine circKLF9 circular RNA m 6 Prediction and Validation of A Modification Sites The mature transcript sequence of circKLF9 was input into SRAMP (Sequence-based RNA Adenosine Methylation Predictor) software for m 6 A-modification site prediction, with parameters set to default values. Results are as follows: Figure 1 As shown, a total of 2 m values ​​with medium confidence were predicted. 6 The A modification sites are located at positions 167 and 181 of the mature transcript, respectively, on adenine. Both sites conform to m 6 The classical conserved motif DRACH (D=A / G / T, R=A / G, A=methylation site, C=C, H=A / C / T) modified with A.

[0033] Example 2: Used for detecting circKLF9 circular RNA m 6 Design and synthesis of a specific primer pair for modifying the core region Regarding the mature bovine circKLF9 transcript m in Example 1 6 A modifies the core area m 6 A modification site information, designed for m 6 A modifier and non-m 6 Two sets of primers for the A-modified sequence can be used to specifically amplify the m of circKLF9. 6 A modifies the core area containing m. 6 A modification site m 6 A modified and unm 6 Sequences of different lengths modified by A are then processed using m-sequences. 6 A. Quantitative analysis of modifications. Based on the GenBank sequence of circKLF9, specific primers for amplifying the mature bovine circKLF9 transcript were created using the NCBI Primer-BLAST primer design platform. 6 A modifies the core area m 6 The primers used for the A modification site are shown in SEQ ID NO. 1-2 (see Table 1) and were synthesized at Shanghai Sangon Biotech Co., Ltd.

[0034] Table 1. Primers for specific detection

[0035] Example 3: Establishment of a MeRIP-qPCR-based method for detecting circKLF9 6 A method to modify state The detection method of this invention is based on m 6 Based on the principle of A-modification-specific PCR, designs targeting m 6 A modifier and non-m 6 Primers with modified sequences (A) can be used for PCR amplification to convert m... 6 A Modification and Non-m 6 A modified DNA sequence is distinguished. This method includes the following steps: 1. Extract RNA from the sample 1.1 Sample Pretreatment Bovine longissimus dorsi muscle sample: Bovine longissimus dorsi muscle tissue with high and low fat content was collected from the area between the 12th and 13th ribs, with a tissue weight of 100 mg. The muscle sample was ground in liquid nitrogen.

[0036] 1.2 RNA extraction ① Add 1 mL of Trizol to the ground tissue for lysis and incubate at room temperature for 3-5 min; ② Centrifuge at 12000 g, 4℃ for 10 min, and transfer the supernatant to a new enzyme-free centrifuge tube; ③ Add 200 μL of chloroform to the lysis buffer at 1 mL of Trizol, vortex vigorously by hand for 15 s, and incubate at room temperature for 3 min; ④ Centrifuge at 13000 g, 4℃ for 10 min; ⑤ Carefully transfer the supernatant (approximately 1 mL, 500 μL / tube) to a new 1.5 mL enzyme-free centrifuge tube; ⑥ Add an equal volume of isopropanol and precipitate at -20℃ for 2 h; ⑦ Centrifuge at 13000 g, 4℃ for 10 min, retain the precipitate, and discard the supernatant; ⑧ Add 1 mL of 75% ethanol, vortex or invert to mix, and centrifuge at 13000 g, 4℃ for 5 min; ⑨ Discard the supernatant, repeat centrifugation and aspirate any remaining liquid, and air dry for 10-15 min; ⑩ Add 50 μL of RNase-free... Dissolve RNA in water.

[0037] 2. RNA quality testing ① Prepare a 1% agarose gel; ② Take 5 μL of RNA sample and electrophoresis at 120 V for 10 min, then take a picture of the gel to check the RNA quality; ③ Store the remaining sample in a -80℃ refrigerator.

[0038] 3. RNA fragmentation method ① Fragment the RNA sample into fragments approximately 300 nt in length by adding 440 μL of fragmentation buffer to the sample and incubating at 94°C for 2 min; ② Add 60 μL of EDTA to stop the fragmentation reaction; ③ Add 2 μL of LGlycogen, 60 μL of sodium acetate, and 1.2 mL of anhydrous ethanol; ④ Incubate at -20°C for 2 h to precipitate the RNA sample; ⑤ Centrifuge at 13000 g and 4°C for 30 min, and discard the supernatant; ⑥ Add 1 mL of pre-cooled 75% ethanol, centrifuge at 13000 g and 4°C for 10 min, discard the supernatant, and allow to air dry at room temperature for 10 min; ⑦ Add 850 μL of IP buffer 1 and 4 μL of RNase inhibitor to dissolve the RNA sample; ⑧ Take 50 μL of RNA sample as the input sample and store it at -80°C for later use; ⑨ Divide the remaining RNA sample into two tubes of 400 μL each, labeled as the IP group and the IgG group.

[0039] 4. Immunoprecipitation Add 20 μL of IP buffer and 2.4 μg of m to the IP set. 6 For the IgG group, 20 μL of IPbuffer2 and 4 μg of IgG Antibody were added, and the mixtures were incubated in a vertical mixer at 4°C for 4 h.

[0040] 5. Prepare protein A / G magnetic beads. ① Take 40 μL of Protein A / G magnetic beads, add 500 μL of IP buffer 3, gently pipette to mix, and place on a magnetic rack to attract the magnetic beads. Remove the supernatant. ② Add 360 μL of IP buffer 3 and 40 μL of BSA to block Protein A / G magnetic beads. Mix and shake at 4℃ for 2 h. ③ Place on a magnetic rack to attract the magnetic beads and remove the supernatant. ④ Add 200 μL of IP buffer 3, gently pipette to mix, and place on a magnetic rack to attract the magnetic beads. Remove the supernatant. Repeat twice. ⑤ Add 200 μL of IP buffer 3, gently pipette to mix, and divide into two tubes of 100 μL each.

[0041] 6. Protein A / G binds to antibodies. ① Add the prepared Protein A / G magnetic beads to the IP group and IgG group, and incubate vertically at 4°C for 1 h; ② Adsorb the magnetic beads onto the magnetic rack and remove the supernatant; ③ Add 500 μL of IP buffer 3 to the IP group and IgG group respectively, wash vertically at 4°C for 5 min, adsorb the magnetic beads onto the magnetic rack, remove the supernatant, and repeat 3 times; ④ Add 200 μL of LELution Buffer and 2 μL of Proteinase K to the IP group and IgG group respectively, digest at 55°C for 30 min and mix vertically; ⑤ Collect the magnetic beads on the magnetic rack and transfer the supernatant to a new RNase-free tube.

[0042] 7. RNA extraction ① Add 150 μL of Elution Buffer to the Input sample; ② Add an equal volume (200 μL) of phenol-chloroform-isoamyl alcohol mixture (25:24:1) to the Input, IP, and IgG samples respectively, and mix by inversion for 15 s; ③ Centrifuge at 13000 g, 4℃ for 10 min, collect the upper aqueous phase, and transfer it to a new RNase-free centrifuge tube; ④ Add 1 μL of Glycogen, 20 μL of Sodium Acetate, and 400 μL of anhydrous ethanol respectively, and mix thoroughly by inversion; ⑤ Incubate at -20℃ or -80℃ for 3 h or overnight to precipitate the RNA sample; ⑥ Centrifuge at 16000 g, 4℃ for 30 min, and discard the supernatant; ⑦ Add 1 mL of pre-cooled 75% ethanol to each sample, centrifuge at 16000 g, 4℃ for 10 min, discard the supernatant, and air dry at room temperature for 10 min; ⑧ Add 20 μL of RNase-free water to each sample and store on ice.

[0043] 8. Testing and Verification ① Take 1 μL of RNA sample and use an Agilent 2100 to detect RNA concentration; ② Refer to the reverse transcription kit instructions to reverse transcribe the RNA sample (IP, IgG, and Input); ③ Prepare the reaction system according to the PCR kit and perform PCR. Take 3-5 μL of PCR product and use 3% agarose gel electrophoresis to detect the amplification effect. The agarose gel electrophoresis image of the extracted RNA is shown in (see...). Figure 2 ); ④ Prepare a 20 μL PCR amplification system according to the instructions of the 2×SYBRGreen qPCR Mix kit, perform qPCR, and then analyze the data to obtain the enrichment efficiency; The PCR reaction system in each PCR tube consisted of: 1 μL each of 10 μM forward and reverse primers, 10 μL of 2×SYBR Premix Ex Taq II, 2 μL of cDNA template, and 6 μL of sterile water.

[0044] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 68℃ annealing / extension for 20 s, for a total of 40 cycles, followed by holding at 95℃ for 10 s after each cycle; the melting curve program was as follows: increase the temperature from 65℃ to 95℃ by 0.5℃ per step and hold for 30 s.

[0045] The results are as follows Figure 2 , Figure 3 As shown, from Figure 2 As can be seen, the 28S and 18S rRNA bands are clear, with no obvious degradation, and the RNA integrity is good; observe the melting curve obtained from PCR amplification ( Figure 3 The results showed a single sharp peak, and a single melting temperature was obtained in each PCR tube, indicating good primer specificity and no non-specific amplification.

[0046] Example 4: circKLF9 m in high-fat and low-fat bovine muscle tissue 6 A Modification and Expression Level Detection Six low-fat bovine muscle tissue samples (Low fat group) and six high-fat bovine muscle tissue samples (High fat group) were collected. The MeRIP-qPCR method established in Example 3 was used to detect the m of circKLF9. 6 Enrichment level was assessed, and the relative expression level of circKLF9 was detected by conventional qPCR. GAPDH was used as the internal reference gene, and the relative expression level was calculated using the 2^(-ΔΔCt) method.

[0047] m 6 The enrichment level detection results are as follows: Figure 4 As shown, the m of circKLF9 in low-fat tissues 6 The enrichment level of A was 1.12±0.15%, while it was 2.35±0.28% in tissues with high fat content; the difference between the two groups was highly significant. p <0.01). The relative expression level of circKLF9 was detected as follows: Figure 5 As shown, the relative expression level of circKLF9 was significantly lower in high-fat tissues than in high-fat tissues (where it was downregulated). p <0.01). The results show that the m of circKLF9 6 The level of A modification is closely related to the fat deposition capacity of bovine muscle tissue. The primers and detection method of this invention can effectively distinguish between muscle tissue samples with high and low fat content, providing a reliable technical means for molecular marker screening of intramuscular fat deposition traits in beef cattle.

[0048] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0049] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this application and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. A circKLF9 m assay for evaluating bovine fat deposition capacity 6 A molecular probe with a modified site, characterized in that... It is an oligonucleotide probe whose nucleotide sequence corresponds to the m-nucleotide sequence of bovine circKLF9 containing adenine at positions 167 and / or 181. 6 The target sequence of the modified site is complementary to that of the target sequence, and the target sequence contains the adapter site of the circKLF9.

2. A circKLF9 m assay for evaluating bovine fat deposition capacity 6 A molecular probe with a modified site, characterized in that... It is a primer pair consisting of a forward primer F and a reverse primer R, and its nucleotide sequence is shown below: F: 5'-CGCTTCATGAGGAGCGACC-3', R: 5'-TTGATCATGCTGGGGTGGAA-3'.

3. A primer-probe combination, characterized in that, It comprises the oligonucleotide probe of claim 1 and the primer pair of claim 2.

4. A reagent kit, characterized in that, It comprises the oligonucleotide probe of claim 1, the primer pair of claim 2, or the primer-probe combination of claim 3, as well as RNase R enzyme and anti-m 6 At least one of the following: antibody A, RNA fragmentation reagent, reverse transcription reagent, and real-time quantitative PCR premix.

5. A bovine circular RNA circKLF9 m 6 A method for detecting A methylation levels, characterized in that... Includes the following steps: (1) Extract total RNA from the bovine samples to be tested; (2) Remove linear RNA from the total RNA of the bovine samples to be tested and enrich circular RNA; (3) The sample enriched with circular RNA is processed into fragmented RNA samples to obtain fragmented RNA samples; (4) Use anti-m 6 Antibody A was used to perform immunoprecipitation on the fragmented RNA sample to obtain immunoprecipitated RNA; (5) The immunoprecipitated RNA was purified and reverse transcribed to obtain cDNA; (6) Using the molecular probe of claim 2 or the primer-probe combination of claim 3, the cDNA is amplified by real-time quantitative PCR; (7) Calculate the m of circKLF9 based on the real-time quantitative PCR amplification results. 6 A. Methylation modification level.

6. The detection method according to claim 5, characterized in that, The bovine sample to be tested was the longissimus dorsi muscle tissue of a cow.

7. The detection method according to claim 5, characterized in that, The reaction system for real-time quantitative PCR amplification in step (6) includes: 1 μL each of forward and reverse primers at a concentration of 10 μM, 10 μL of 2×SYBR PremixEx Taq II or PCR premix containing fluorescently labeled probes, 2 μL of cDNA template and 6 μL of sterile water.

8. The detection method according to claim 5, characterized in that, The reaction procedure for real-time quantitative PCR amplification in step (6) is as follows: pre-denaturation at 95℃ for 5 min; followed by 40 cycles, each cycle including denaturation at 95℃ for 10 s and annealing extension at 68℃ for 20 s; If the oligonucleotide probe method is used, the fluorescence signal is collected during the annealing extension stage.

9. The use of the molecular probe of claim 1 or 2, the primer-probe combination of claim 3, or the kit of claim 4 in the preparation of products for detecting and evaluating bovine fat deposition capacity or intramuscular fat content.

10. The application according to claim 9, characterized in that, The product was used to detect the m of circKLF9 in the sample to be tested. 6 A modification level and / or expression level were used to assess intramuscular fat content in cattle; among which, the m of circKLF9 was... 6 The level of A modification was positively correlated with the fat deposition capacity of cattle, and / or, the expression level of circKLF9 was negatively correlated with the fat deposition capacity of cattle.

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