Application of MYLK gene and CPSF2 gene in regulation and control of intramuscular fat deposition of beef

Abstract

The invention discloses application of an MYLK gene and a CPSF2 gene in regulation and control of beef intramuscular fat deposition, and belongs to the technical field of gene engineering. By studying an APA mechanism in a bovine intramuscular fat deposition process, it is found that in an IMF deposition process, 3 'UTR of an MYLK gene is shortened, the expression quantity of the MYLK gene is increased, miRNA participating in IMF deposition is deleted in the shortening process, and then IMF deposition is regulated to be reduced; after the 3 'UTR of the CPSF2 gene is shortened, expression up-regulation also occurs, and IMF deposition is regulated and controlled to be reduced. Research results of the invention provide new theoretical support and technical guidance for beef quality prediction and high-quality beef cattle breeding, and have important meanings for promoting animal husbandry development and improving animal husbandry production benefits.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of MYLK gene and CPSF2 gene in regulating beef intramuscular fat deposition. Background Art

[0002] In recent years, with the improvement of people's living standards, the requirements for beef quality have become more and more stringent. Among them, the process of intramuscular fat (IMF) deposition is crucial to the formation of meat quality and the improvement of taste. The intramuscular fat content affects the shear force, flavor, juiciness and tenderness of the muscle. The flavor is closely related to the fat and fat-soluble substances in the muscle. As the main source of volatile compounds, intramuscular fat is considered to be a species-specific flavor precursor. The types and proportions of fatty acids in intramuscular fat have a great influence on flavor substances. The differences in flavors of different meats are due to their different oxidation products. The unsaturated fatty acids in intramuscular fat are essential fatty acids for the human body and have high nutritional value. IMF exists in the epimysium, perimysium and endomysium of muscle fibers. The deposition of IMF can promote the separation of muscle fiber bundles, thereby improving muscle tenderness. Studies by Zuo Xiuli and others have shown that the marbling grade has a very significant positive effect on the tenderness of beef. Research by Mao Yanwei and others showed that IMF can increase the water-binding capacity of muscle, reduce drip loss and cooking loss of meat, and improve meat quality; in addition, IMF is negatively correlated with muscle shear force, but positively correlated with muscle pH value.

[0003] Factors that affect intramuscular fat deposition in beef cattle include genetic factors, management factors, and nutritional factors. Among them, IMF deposition can be regulated by specific genes (PPARγ, C / EBPα, ZFP423, and FABP4, etc.) and signaling pathways (AMPK, WNT, and MAPK, etc.). In addition, epigenetic modifications are also involved in the various biological processes of IMF deposition. However, in addition to transcriptional regulation and epigenetic regulation, post-transcriptional regulation also plays a vital role in the process of IMF deposition.

[0004] Alternative polyadenylation (APA) is a post-transcriptional regulatory process that generates multiple transcripts with different 3' untranslated region (3'UTR) lengths by selectively shearing and adding polyadenylic acid tails of different lengths. It plays an important role in gene expression regulation, affecting transcript stability, translation efficiency, and functional diversity. Current studies have shown that changes in 3'UTR length can affect gene translation efficiency, and the effect of APA on translation efficiency is mainly regulated by changing the availability of miRNA binding sites. APA is widely present in mammals and plays a key role in cell development, biological processes, and disease progression.

[0005] Recent studies have found that APA is associated with IMF deposition and is involved in regulating muscle development and adipogenic differentiation. APA can mediate miRNA to regulate muscle stem cell function. Pax3 is an important myogenic regulator. During the development of myogenic satellite cells, Pax3 transcripts are affected by APA, resulting in the shortening of the 3′UTR of the transcript, making it unable to be regulated by miR-206. In mouse myoblasts, RBFOX2 regulates the mRNA expression levels of contractile genes and mitochondrial genes through APA. In the study of the fast and slow skeletal muscle transcriptomes of pigs, it was found that APA may play a role in the development of fast and slow muscles under the regulation of miRNAs and RNA-binding proteins. In 3T3-L1 preadipocytes, HO1 inhibits preadipocyte differentiation, while the HO1 3′UTR short variant produced by APA escapes the inhibitory effect of miRNA, resulting in a higher expression level of HO1, thereby having a stronger inhibitory effect on preadipocyte differentiation. However, studies on the mechanism of APA during bovine IMF deposition are rarely reported. Summary of the invention

[0006] The purpose of the present invention is to provide an application of MYLK gene and CPSF2 gene in regulating beef intramuscular fat deposition to solve the problems existing in the above-mentioned prior art. The present invention studies the APA mechanism in the process of intramuscular fat deposition in cattle and finds that MYLK gene and CPSF2 gene have the function of regulating fat deposition, which provides new theoretical support for the prediction of beef quality and the breeding of high-quality beef cattle.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides application of MYLK gene in regulating beef intramuscular fat deposition.

[0009] The present invention also provides application of CPSF2 gene in regulating beef intramuscular fat deposition.

[0010] The present invention also provides application of a reagent for detecting the expression amount of the MYLK gene in predicting or assisting in predicting beef quality.

[0011] The present invention also provides application of a reagent for detecting the expression amount of the MYLK gene in breeding beef cattle with high-quality beef quality.

[0012] The present invention also provides application of a reagent for detecting the expression amount of the CPSF2 gene in predicting or assisting in predicting beef quality.

[0013] The present invention also provides application of a reagent for detecting the expression amount of the CPSF2 gene in breeding beef cattle with high-quality beef quality.

[0014] The present invention discloses the following technical effects:

[0015] The present invention studies the APA mechanism in the process of intramuscular fat deposition in cattle, establishes the APA map in the process of IMF deposition, and finds that the shortening of 3′UTR is related to IMF deposition, which can lead to an increase in gene expression levels through the loss of miRNA binding sites. Further studies have found that during the IMF deposition process, the 3′UTR of the MYLK gene is shortened, and the expression level of the MYLK gene is increased. During the shortening process, bta-miR-206, bta-miR-32 and bta-miR-195 involved in IMF deposition are lost, regulating the reduction of IMF deposition; after the 3′UTR of the CPSF2 gene is shortened, the expression is also upregulated, regulating the reduction of IMF deposition. The research results of the present invention provide new theoretical support and technical guidance for the prediction of beef quality and the breeding of high-quality beef cattle, which is of great significance to promoting the development of animal husbandry and improving the production efficiency of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The results of the comprehensive analysis of RNA-seq data; A is the PDUI score map of each gene in the H and L groups, the dotted line represents the threshold of 0.1, the blue dots represent genes with extended 3'UTR, and the red dots represent genes with shortened 3'UTR; B is the volcano map of A, representing |δPDUI|>0.1 (FDR<0.05) of genes with shortened (red) and extended (blue) 3'UTRs; C is a comparison of the 3'UTR lengths between the transcripts that were significantly changed in the H group and other transcripts that did not exceed the threshold detected in the H and L group samples; D is a bar graph of the number of base pairs lost / gained by genes with changed 3'UTRs;

[0018] Figure 2 is the relationship between 3′UTR length and aUTR length;

[0019] Figure 3 Figure 2 is the detection result of related gene expression in APA-driven IMF deposition; A is the log2-fold change of gene expression with 3'UTR changes; the up-regulated genes (red dots) and down-regulated genes (blue dots) on the left represent genes with shortened 3'UTR, and the right represents genes with extended 3'UTR; B is a comparison of the number of genes with up-regulated (red) or down-regulated (blue) 3'UTR changes in IMF;

[0020] Figure 4 The results of the establishment and analysis of the miRNA-APA network during fat deposition; A is a distribution bar graph of miRNA binding sites lost due to 3'UTR shortening; B is the miRNA-APA network, where the lines represent miRNAs affected by APA genes and gene-gene, and the line PPI>0.7; C is the regulatory pattern of APA and MYLK 3'UTR with conserved miRNA site locations;

[0021] Figure 5 is the expression of MYLK gene in group H and group L;

[0022] Figure 6 Figure 2 shows the expression of CPSF2 gene in the H group and the L group. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Methods

[0030] 1.1 Sample collection and IMF content determination

[0031] Thirty crossbred Wagyu cattle (Qinchuan cattle × Wagyu cattle) in good health, similar age and weight were selected and raised together. These cattle were fed under the same nutritional level and management conditions. Before slaughter, cattle needed to fast for 24 hours, but they could drink water freely. After slaughter according to the standard process, the longissimus dorsi muscle was collected and frozen in liquid nitrogen, and then stored at -80℃ for RNA extraction. At the same time, about 500g of the longissimus dorsi muscle was collected for the determination of IMF content. The IMF content of the longissimus dorsi muscle was determined by Soxhlet extraction. Three individuals with high IMF content (H) (32.96±3.26%) and three individuals with low IMF content (L) (10.91±1.85%) were selected from the 30 cattle for further analysis. The IMF content of these individuals was significantly different (P<0.05).

[0032] 1.2 RNA preparation, library construction and sequencing

[0033] Total RNA was extracted using the Trizol kit (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed on an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase-free agarose gel electrophoresis. After total RNA extraction, rRNA was removed and mRNA and ncRNAs were retained. The enriched mRNAs and ncRNAs were fragmented into short fragments using a fragmentation buffer and reverse transcribed into cDNA using random primers. The second-strand cDNA was synthesized by DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP) and buffer. Next, the cDNA fragments were purified using a QiaQuick PCR extraction kit (Qiagen, Venlo, the Netherlands), the ends were repaired, poly (A) was added, and ligated to the Illumina sequencing adapter. The second-strand cDNA was then digested with UNG (uracil-N-glycosylase). The digested products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using an Illumina HiSeqTM 4000.

[0034] 1.3 APA analysis

[0035] Sequence files were quality controlled using FastQC v0.11.9 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ), and low-quality reads were trimmed using Trim Galore v0.6.0 (https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ). All sequence files were aligned to the bovine reference genome ARS-UCD1.2 using HISAT2 v2.2.1. BAM files of the six samples were converted to wig files using bedtoolsgenomecov v2.30.0. The wig files were processed using DaPars v1.0.0 (https: / / github.com / ZhengXia / DaPars) to identify differences in 3′UTRs between samples with high and low IMF content. Proximal polyA sites were predicted and the percentage of distal polyA site usage index (PDUI) was calculated. PDUI values ​​range from 0 to 1. Higher PDUI scores represent higher utilization rates of distal PAS sites.

[0036] To compare the 3′UTR changes of specific genes in high and low IMF content samples, the PDUI scores of high IMF content samples (MeanPDUIH) and low IMF content samples (MeanPDUIL) were averaged. The mean PDUI score change between high and low IMF content samples was calculated for each gene (ΔPDUI = MeanPDUIH - MeanPDUIL) as a measure of 3′UTR differences. The significance of this difference was assessed by the algorithm Fisher's exact test and further adjusted using Benjamini-Hochberg (BH) to control the false discovery rate, using 0.05 as a threshold. Genes with significant changes in 3′UTR landscape (padj ≤ 0.05) were retrieved and then gene enrichment analysis was performed using DAVID v6.8 (https: / / david.ncifcrf.gov / ).

[0037] 1.4 Differential gene expression analysis

[0038] The raw count matrices of the six samples were generated using StringTie v2.1.7 and converted into transcripts per million (TPM) matrices. The R package DESeq2 v1.20 was used to detect differentially expressed genes (DEGs), and the threshold for screening differential genes was |log2FC|≥0.585. The p value was corrected using Benjamini-Hochberg to obtain FDR, with FDR<0.05 as the threshold.

[0039] 1.5 Construction of miRNA-APA network

[0040] The raw count matrices of miRNA expression of the six samples were normalized by transcripts per million (TPM), and miRNAs with an average TPM ≥ 100 were defined as highly expressed miRNAs. The miRNA binding site information was downloaded from TargetScanHuman 7.2, and then the binding sites of highly expressed miRNAs were mapped in the aUTR regions of differential APA events. PPI network analysis of differential APA event-related genes was performed using STRING v11.5, and gene pairs with interaction scores greater than 0.7 were retained. Finally, miRNA-APA was merged with PPI, and the miRNA-APA network was visualized using Cytoscape v3.10.0.

[0041] 1.6 Integrated genomics viewer

[0042] IGV v2.14.0 was used to display the length and abundance information of 3′UTR sequences in samples with high and low IMF content. The reference genome (Bos_taurus.ARS-UCD1.2.dna.toplevel.fa) and annotation files (Bos_taurus.ARS-UCD1.2.107.gtf) were downloaded from the Ensembl database and imported into IGV. The Bam files of the six samples were then imported into IGV to view the length and sequence differences of the 3′UTR.

[0043] 2. Results

[0044] 2.1 APA events during fat deposition

[0045] To explore APA changes during bovine IMF deposition, RNA-seq data from six samples (three longissimus dorsi muscles with high and three longissimus dorsi muscles with low IMF content) were analyzed by DaPars. To compare the 3′UTR changes of a gene between samples with high IMF content (H) and low IMF content (L), the PDUI score of the gene was averaged in samples with high IMF content (MeanPDUIH) and low IMF content (MeanPDUIL). The change in the mean PDUI score between H and L samples for each gene was calculated (ΔPDUI=MeanPDUIH-MeanPDUIL) and used as a measure of 3′UTR shortening or lengthening events associated with fat deposition. After strict screening (adjusted p-value<0.05; |MeanPDUIH-MeanPDUIL|>0.1, Figure 1 A and B), 363 differential APA events were obtained, and 300 genes were identified to have significantly different 3′UTRs. Between H and L longissimus dorsi muscles, 175 genes showed 3′UTR shortening (ΔPDUI=MeanPDUIH-MeanPDUIL≥0.1), while 125 genes showed 3′UTR elongation (ΔPDUI=MeanPDUIH-MeanPDUIL<-0.1). It is worth noting that the number of shortened 3′UTR events exceeded the number of elongated 3′UTR events. Transcripts with significant changes were observed to show longer 3′UTR lengths than transcripts below the threshold ( Figure 1 The lengths of IMF deposition-related shortening and elongation events were mainly 200-300 bp and >500 bp, respectively ( Figure 1 The length of the variable 3′UTR (aUTR) is defined as the distance between the distal polyA site and the proximal polyA site. The results showed that the aUTR length of most APA events was 200-300 nt. At the same time, as the 3′UTR lengthened, the aUTR also lengthened (R 2 =0.79)( Figure 2 These findings suggest that genes with longer 3′UTRs are more likely to be regulated by APA during IMF deposition.

[0046] 2.2 Changes in APA-driven gene expression

[0047] To determine whether APA events drive changes in gene expression in fat deposition, we investigated the association between specific APA events and changes in gene expression by calculating differential gene expression between tissues with high and low IMF content (H and L). Figure 3Among the genes with shortened 3′UTR, 16 genes were significantly up-regulated and 18 genes were significantly down-regulated. Similarly, among the genes with extended 3′UTR, 10 genes were significantly up-regulated and 11 genes were significantly down-regulated ( Figure 3 (B) The increased expression of 16 genes may be the result of genes with shortened 3′UTRs escaping miRNA inhibition. However, compared with all DEGs, the upregulation of genes with shortened 3′UTRs was not significant, suggesting that there are other regulatory mechanisms for gene expression. In addition, three genes with extended 3′UTRs, ACVR2B, LMOD3, and RTN4, were upregulated during IMF deposition, and these three genes are associated with myocyte proliferation and differentiation. Previous studies have shown that the 3′UTR isoforms of some genes can increase their expression through trans-regulatory mechanisms.

[0048] 2.3 miRNA-APA network in fat deposition

[0049] To verify that the 3′UTR shortened by APA during IMF deposition may escape the inhibition of miRNA and thus increase gene expression, the distribution of miRNA binding sites lost due to shortened 3′UTR length was calculated. This analysis showed that 44.1% of the genes lost at least one highly conserved miRNA binding site ( Figure 4 This indicates that the change of miRNA binding sites is a common regulatory mode mediated by APA.

[0050] To further analyze the changes in miRNA binding sites that may be caused by APA events during IMF deposition, miRNA expression data of 6 samples were collected, and the miRNA expression was normalized using TPM values. Highly expressed miRNAs with TPM ≥ 100 were selected, and then these miRNA binding sites were aligned to the aUTRs of differential APA events. In this way, a total of 146 IMF-related important miRNA sites were found that may be gained or lost due to 363 differential APA events. Afterwards, STRING was used to identify protein-protein interactions (PPIs) of APA events. These results were then used to construct a miRNA-APA network ( Figure 4 B), among the top 10 miRNAs regulating this network, bta-miR-32 has been shown to regulate fat deposition. Bta-miR-206, bta-miR-195, and bta-miR-16a have been reported to be associated with the proliferation and differentiation of muscle cells, so they may also be involved in regulating the deposition of IMF. In this regulatory network, an important gene MYLK was also found. When the 3′UTR of MYLK is shortened, the miRNA binding sites of bta-miR-32, bta-miR-206, and bta-miR-195 are lost ( Figure 4Therefore, MYLK may be a key gene in the IMF deposition process.

[0051] In addition, a key regulatory factor regulating APA events, CPSF2, was discovered. During IMF deposition, CPSF2 gene expression was upregulated after its 3′UTR was shortened. Cleavage and Polyadenylation Specificity Factor Subunit 2 (CPSF2) is a member of the cytoplasmic polyadenylation enzyme (CPF) family and a gene that can recognize polyadenylation signals. CPSF2 can recognize AAUAAA polyadenylation signals and plays an important role in nuclear export, translation initiation, and transcript stability. CPSF2 may be a target molecule of intronic miRNA. When CPSF2 is silenced, polyadenylation tends to recognize typical poly(A) signals, and the 3′UTR of most genes is elongated. In cancer cells, CPSF2 shortens the 3′UTR of glycolysis-related genes by regulating APA events, thereby promoting cancer cell growth by upregulating the glycolysis pathway. In addition, during the development of cancer cells, the increased expression of mRNA 3′ end processing factors (including CPSF2) leads to the shortening of mRNA 3′UTR. Therefore, it is believed that CPSF2 may be a key regulator in the IMF deposition process. The miRNA-APA regulatory network of MYLK and CPSF2 is shown in Table 1.

[0052] Table 1 miRNA-APA regulatory network constructed using STRING and TargetScanHuman

[0053]

[0054]

[0055] Example 2 Differential expression of MYLK and CPSF2 genes

[0056] 1. Design and synthesis of primers

[0057] Primer Premier5 software was used to design primers based on the cDNA sequences of bovine MYLK, CPSF2 and GAPDH genes in Genebank. The primer sequences of each gene are shown in Table 2.

[0058] Table 2 Primer sequences

[0059]

[0060] 2. RNA Extraction

[0061] (1) The bovine intramuscular adipose tissue of the individuals in Group H and Group L determined in Example 1 was taken out of a -80°C refrigerator and placed on ice. After it was completely dissolved, the tissue block was taken out from the RNAsolid tissue RNA stabilization storage solution with clean tweezers, and then the surface liquid was absorbed with clean absorbent paper and placed in a 1.5 ml EP tube.

[0062] (2) Add 1 ml of Lysis Buffer RZ for every 50-100 mg of tissue and homogenize using a homogenizer.

[0063] (3) Place the homogenized sample at 15-30°C for 5 min to allow the nucleic acid-protein complex to be completely separated.

[0064] (4) Centrifuge at 4°C, 12,000 rpm for 5 min and transfer the supernatant into an RNase-free centrifuge tube.

[0065] (5) Add 200 μL of chloroform and cover the centrifuge column tightly. Shake vigorously for 15 seconds and let stand at room temperature for 3 minutes.

[0066] (6) Centrifuge at 4°C and 12,000 rpm. The sample in the centrifuge column is separated into an upper colorless aqueous phase containing RNA, an intermediate layer, and a lower yellow organic phase. The volume of the aqueous phase is about half the volume of RZ. Use a pipette to carefully transfer the upper aqueous phase to a new centrifuge tube.

[0067] (7) Add 200 μL of anhydrous ethanol and shake to mix, and transfer the resulting precipitate and liquid to the adsorption column CR3. Centrifuge at 12,000 rpm for 30 s at 4°C, and then discard the waste liquid in the collection tube below the adsorption column.

[0068] (8) Add 500 μL of deproteinized solution RD to the adsorption column CR3. Centrifuge at 4°C, 12,000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0069] (9) Add 500 μL of rinse solution RW to the adsorption column CR3 and let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 30 s at 4°C and discard the waste liquid in the collection tube.

[0070] (10) Repeat step (9) to ensure that the impurities are completely rinsed.

[0071] (11) Transfer the adsorption column CR3 to a new 1.5 mL centrifuge tube and add 30 μL RNase-Free ddH2O. Incubate at room temperature for 5 min and centrifuge at 12,000 rpm for 2 min at 4 °C.

[0072] (12) Use Nanodrop to measure RNA concentration and control OD 260 / OD 280 Approximately equal to 2.

[0073] (13) RNA gel electrophoresis was used to verify RNA integrity.

[0074] 3. cDNA Preparation

[0075] Follow the manufacturer's instructions for reverse transcription kits. Use eight rows in a row, add 4μl of 5× PrimeScript RT Master Mix, 1000ng RNA, and adjust to 20μl with DEPC water to each well. Mix well using a centrifuge. Place in a PCR instrument, reverse transcribe at 37℃ for 15min, heat inactivate reverse transcriptase at 85℃ for 5s, cool at 4℃, and store at -20℃ for later use.

[0076] 4. qRT-PCR reaction

[0077] The specific operation steps of qRT-PCR and the amount of each reagent added are shown in Table 3.

[0078] Table 3 Reagents and dosages required for qRT-PCR

[0079]

[0080]

[0081] Add the prepared reaction solutions of each dose group into eight tubes, mix and centrifuge, and place in a fluorescence quantitative PCR instrument. The reaction setting program is shown in Table 4.

[0082] Table 4 PCR reaction program

[0083]

[0084] GAPDH gene was used as the internal reference, and the relative content of the target gene amplification product was expressed as 2 -ΔΔCt The specific calculation method is as follows: First, use the Ct value of the target gene - the Ct value of the internal reference gene: ΔCT = CT (target gene) - CT (internal reference); secondly, use the ΔCt value of the treated sample - the ΔCt value of the control sample: ΔΔCT = ΔCT (experimental sample) - ΔCT (control sample), and use any sample that can represent one-fold the expression level of the target gene as the control sample; finally, calculate the expression level ratio: 2 -ΔΔCt =Ratio of relative gene expression levels.

[0085] The expression of MYLK and CPSF2 genes in the H and L groups is shown in Figure 2 Figure 5 and Figure 6 As shown, it can be seen that the expression of MYLK and CPSF2 genes in the two groups is significantly different. Combined with the above results, MYLK and CPSF2 genes are important genes regulating IMF deposition.

[0086] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of MYLK gene in regulating beef intramuscular fat deposition.

2. Application of CPSF2 gene in regulating beef intramuscular fat deposition.

3. Application of reagents for detecting MYLK gene expression in predicting or assisting in predicting beef quality.

4. Application of reagents for detecting MYLK gene expression in breeding beef cattle with high-quality beef.

5. Application of reagents for detecting CPSF2 gene expression in predicting or assisting in predicting beef quality.

6. Application of reagents for detecting CPSF2 gene expression in breeding beef cattle with high-quality beef.

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