Primer group for detecting methylation level of intron 1 of goat fat deposition related gene ACSL5 and application of primer group
By designing a primer set to detect DNA methylation in the intron 1 region of the goat ACSL5 gene, the problem of the existing technology failing to effectively detect the methylation level of genes related to intramuscular fat deposition in goats was solved, and the accurate judgment of the intramuscular fat content of the longissimus dorsi muscle of goats was achieved, which promoted the improvement of growth traits of high IMF varieties.
Patent Information
- Application Number
- CN202511209134.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-27
- Publication Date
- 2025-10-10
AI Technical Summary
Existing technologies fail to effectively detect and regulate the DNA methylation level of intron 1 of the gene ACSL5 related to intramuscular fat deposition in goats, affecting the improvement of growth traits of high-IMF breed goats.
A specific primer set was designed and used to detect DNA methylation in the intron 1 region of the goat fat deposition-related gene ACSL5, including the upstream outer primer CpG1-OF, the downstream outer primer CpG1-OR, the upstream inner primer CpG1-IF, and the downstream inner primer CpG1-IR. The methylation level was analyzed by PCR amplification and sequencing.
A comprehensive and accurate detection of the CpG island methylation level in the intron 1 region of the goat ACSL5 gene was achieved, which assisted in determining the intramuscular fat content of the longissimus dorsi muscle of the goat and improved the accuracy and precision of the detection.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a primer set for detecting the methylation level of intron 1 of the fat deposition-related gene ACSL5 in goats and an application thereof. Background Art
[0002] As Chinese consumers' demand for high-quality, flavorful meat continues to rise, goat meat is becoming a staple in their daily diet, and the demand for high-quality goat meat is also increasing. Therefore, it is of great significance to study the molecular regulatory mechanisms that improve the growth traits of local goat breeds, identify, and fully utilize the major genes that regulate intramuscular fat deposition in goats, and combine this with traditional breeding methods to improve goat growth traits.
[0003] DNA methylation is an epigenetic modification mediated by the DNA methyltransferase (DNMT) family. Its molecular basis is to catalyze the methylation of the fifth carbon atom of cytosine. Although methylation does not alter the primary structure of DNA, it nonetheless regulates gene expression. This reaction uses S-adenosylmethionine as a methyl donor. Through covalent binding mediated by the DNMT active center, the methyl group is specifically transferred to the cytosine residue in CpG dinucleotides, forming the 5-methylcytosine (5mC) epigenetic mark. 5mC is a widespread DNA modification. Certain locations in the genome have a high density of CG sequences (GC content ≥50%), known as CpG islands. These sites exhibit a non-random distribution across the genome, with approximately 70% of CpG islands located in gene promoters, the first exon, and the first intron, constituting key epigenetic switches for transcriptional regulation. In goats, DNA methylation is primarily concentrated at CpG dinucleotide sites, forming mCpGs. However, only 60% to 90% of CpG sites in the genome are methylated, and this dynamic modification process is strictly regulated by the cell cycle. Methylation of CpG islands in promoters or first introns may lead to transcriptional silencing of target genes.
[0004] Intramuscular fat (IMF) content is the most critical indicator of goat meat quality, and this trait is regulated by multiple genes with minimal effects. Previous studies have found that the IMF content of Fuqing goats (3.69%) is significantly higher than that of Nubian goats (1.83%) (P<0.01). The project team conducted a combined analysis of whole-genome methylation resequencing and transcriptome sequencing in Fuqing and Nubian goats, and screened for a key candidate gene significantly associated with goat IMF content, long-chain fatty acyl-CoA synthetase 5 (ACSL5). The ACSL5 gene is a widely expressed gene, but its primary target organ is the liver. The ACSL5 gene is a key activator of dietary long-chain fatty acids and can reduce the activation level of fatty acids in the human jejunum. Studies have found that overexpression of the ACSL5 gene can promote fatty acid oxidation and free radical formation, and can downregulate insulin signaling in human skeletal myotubes. Knocking out the ACSL5 gene reduces the synthesis of neutral lipids secreted by the liver, and the activity of the ACSL5 gene may affect intestinal microbial relationships and, further, lipid metabolism. Goats derive 80% of their energy from volatile fatty acids, and the ACSL5 gene plays a crucial role in fatty acid and energy metabolism. Therefore, the ACSL5 gene has a significant impact on IMF traits in goats. However, the impact of DNA methylation levels in intron 1 of the ACSL5 gene on IMF content at different stages in Fuqing goats, a high-IMF breed, remains unknown. Furthermore, the mechanism by which DNA methylation modifies IMF traits in goats and the corresponding detection methods are still unknown. Summary of the Invention
[0005] The purpose of the present invention is to provide a primer set for detecting the methylation level of intron 1 of the goat fat deposition-related gene ACSL5 and an application thereof.
[0006] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a primer set for detecting the DNA methylation level of the first intron of the goat fat deposition-related gene ACSL5, wherein the primer set has the following sequence: Upstream outer primer CpG1-OF: 5'-GAGTGTTTGATTTTGGAGGAAATTATT-3', Downstream outer primer CpG1-OR: 5'-CTAATCCCTTAACCTCATAAAATCCCA-3', Upstream inner primer CpG1-IF: 5'-TATTTTTTTTAGGTTTTAAGTTTTT-3', Downstream inner primer CpG1-IR: 5′-CCATCTTTAAATTCATCTCTTAACC-3′.
[0007] The second aspect of the present invention provides the use of the above primer set in preparing a kit for detecting the DNA methylation level of the first intron of the fat deposition-related gene ACSL5 in goats.
[0008] A third aspect of the present invention provides a kit for detecting the DNA methylation level of the first intron of the fat deposition-related gene ACSL5 in goats, the kit comprising the above-mentioned primer set.
[0009] Furthermore, the method for using the above kit comprises the following steps: S1: Extract the genomic DNA of the goat to be tested and treat it with bisulfite; S2: Using bisulfite-treated genomic DNA as a template, PCR amplification was performed using the upstream outer primer CpG1-OF and the downstream outer primer CpG1-OR to obtain PCR product 1; S3: Using PCR product 1 as a template, PCR amplification was performed using the upstream inner primer CpG1-IF and the downstream inner primer CpG1-IR to obtain PCR product 2; S4: Sequencing the PCR product 2, analyzing the sequencing results, and determining the DNA methylation level of the first intron of the fat deposition-related gene ACSL5 in the goat to be tested; The genomic DNA is extracted from brain tissue, liver tissue, kidney tissue, adipose tissue, longissimus dorsi muscle tissue or biceps femoris muscle tissue of the goat to be tested; The reaction system of the PCR amplification was as follows: 1 μL of 50 ng / μL template, 0.5 μL of 10 pmol upstream and downstream primers, 12.5 μL of 2×Premix Taq Mix, and 10.5 μL of deionized water.
[0010] The reaction program of the PCR amplification is: 94°C for 5 min; 94°C for 30 s, 58-67°C for 50 s, 40 cycles; 72°C for 30 s; 72°C for 5 min.
[0011] A fourth aspect of the present invention provides the use of the above primer set and kit in preparing a product for assisting in determining the intramuscular fat content of the longissimus dorsi muscle of a goat; Furthermore, the corresponding relationship between the DNA methylation level of the first intron of the fat deposition-related gene ACSL5 in goat liver tissue and the intramuscular fat content of the longissimus dorsi muscle of the goat was used to assist in judging the level of intramuscular fat content of the longissimus dorsi muscle of the goat to be tested. The DNA methylation level of the first intron of the fat deposition-related gene ACSL5 in goat liver tissue was significantly negatively correlated with the intramuscular fat content of the longissimus dorsi muscle of the goat.
[0012] The significant advantages of the present invention are: The primer set and method of the present invention can detect the methylation level of the CpG island in the intron 1 region of the goat ACSL5 gene, covering all CpG sites in the intron 1 region of the gene, including statistical analysis of regional methylation and individual sites in the region, thereby improving the accuracy of methylation detection. The primer set and method of the present invention can also detect the methylation level of all CpG sites in the intron 1 region of the ACSL5 gene in tissue samples of goats of different ages, and are not limited to the methylation of CpG sites. It has the advantages of high accuracy, providing a relatively comprehensive and accurate detection method for methylation detection in the intron 1 region of the goat ACSL5 gene, and can be used to assist in the determination of intramuscular fat content of the longissimus dorsi muscle of goats. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 The figure shows the predicted CpG island of the first intron of goat ACSL5 gene and the electrophoresis pattern of the fragment (SEQ ID NO.1).
[0014] Figure 2 The results of CpG island sequencing (forward) and CpG site analysis of the first intron of the goat ACSL5 gene are shown. The CpG sites are in the boxes.
[0015] Figure 3 This is the methylation map of the CpG island in the first intron of the goat ACSL5 gene. The bar graph shows the methylation rates of various tissues in Fuqing goats during the three critical periods.
[0016] Figure 4 This is the intramuscular fat (IMF) content of the longissimus dorsi muscle of Fuqing goats at birth, weaning and adult stages. DETAILED DESCRIPTION
[0017] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
[0018] Example 1: 1 Animals and sample collection Nine Fuqing goats were used in the experiment, including three newborn male lambs, three weaned goats and three adult goats. They were raised and slaughtered at the breeding farm of Fujian Academy of Agricultural Sciences. Immediately after slaughter, six tissue samples, including brain, liver, kidney, fat, longissimus dorsi and biceps femoris, were collected from the nine goats. The samples were then quickly placed in liquid nitrogen and subsequently transferred to a -80℃ freezer.
[0019] 2 Determination of intramuscular fat content in goats at different stages Longissimus dorsi muscle tissue from nine Fuqing goats was minced and placed in a crucible. The minced pieces were then oven-dried at 105°C for 6 hours. The dried samples were removed, crushed using a grinder, and passed through a 40-mesh sieve. After impurities were removed, the samples were placed in a ziplock bag and stored in a desiccator until ready for use. Intramuscular fat (IMF) content was determined using Soxhlet extraction (GB / T 5009.6-2003).
[0020] 3DNA extraction and bisulfite treatment Genomic DNA was extracted from six tissue samples using the phenol-based method and stored at −80°C. The genomic DNA was bisulfite treated strictly according to the instructions of the EpiTect Bisulfite Kit (QIAGEN, Germany).
[0021] 4PCR reaction system and amplification procedure A BSP nested PCR primer set was designed for the CpG island in intron 1 of the goat fat deposition-related gene ACSL5 (GenBank: NC_030833.1): upstream outer primer CpG1-OF, downstream outer primer CpG1-OR, upstream inner primer CpG1-IF, and downstream inner primer CpG1-IR.
[0022] Using bisulfite-treated genomic DNA as a template, PCR amplification was performed using the upstream outer primer CpG1-OF and the downstream outer primer CpG1-OR to obtain PCR product 1. Using PCR product 1 as a template, PCR amplification was performed using the upstream inner primer CpG1-IF and the downstream inner primer CpG1-IR to obtain PCR product 2. The PCR amplification reaction system consisted of 1 μL of 50 ng / μL template, 0.5 μL each of 10 pmol upstream and downstream primers, 12.5 μL of 2× Premix Taq Mix, and 10.5 μL of deionized water. The PCR amplification reaction program was as follows: 94°C for 5 min, 40 cycles of 94°C for 30 s, 58-67°C for 50 s, 72°C for 30 s, and 72°C for 5 min.
[0023] Table 1 Primer sequence information 5. Total RNA extraction, cDNA synthesis and cDNA pool construction Total RNA of all tissue samples was extracted using an RNA extraction kit and RNase-free DNase I (TAKARA, Japan), and DNA impurities were removed using RNase-free DNase I. The quality of the total RNA was detected using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, USA) and 1% agarose gel electrophoresis. According to the kit instructions, the total RNA was reversely transcribed into cDNA using a PrimeScript RT reagent Kit (TAKARA, Japan). TM RT reagent Kit (TAKARA, Japan) to reverse the total RNA into cDNA.
[0024] 6PCR product purification, ligation, transformation, and monoclonal screening PCR product 2 was subjected to 0.8% agarose gel electrophoresis detection, and the target fragment was purified and recovered according to the EasyPure Quick Gel Extraction Kit (Quanjing, China) and its quality was detected. After passing the quality detection, it was ligated with T vector (Promega, USA) at 4°C overnight. The ligation system is shown in Table 2.
[0025] Table 2 Ligation system The ligation product was added to 50 μL Trans1-T1 competent cells, mixed gently, and then subjected to ice bath for 30 min, 42°C water bath heat shock for 30 s, immediately placed on ice for 2 min, added with 200 μL LB liquid medium, and placed in a 37°C constant temperature shaker for 1 h. Then, centrifuged at 2000 rpm for 30 s, discarded part of the supernatant, suspended the bacterial body, and evenly coated the whole bacterial liquid on LB solid medium with ampicillin resistance, and cultured at 37°C overnight. The next day, 20 single colony clones were selected for bacterial liquid PCR, and 15 positive clones were selected from each sample for sequencing in Shanghai Shengong Biological Engineering Co., Ltd.
[0026] 7Sequence alignment and methylation pattern analysis The sequencing results of the target CpG island were analyzed. By comparing with the original sequence (GenBank: NC_030833.1), if the CpG site in the sequencing sequence remains unchanged as CG, it indicates that the CpG site in the genome is methylated. If the CpG becomes TG, it indicates that the site in the genome is not methylated. Through this method, the methylation rate of ACSL5 gene intron 1 CpG island (the overall methylation rate of CpG island and the methylation rate of each CG site) in each sample was determined.
[0027] 8Methylation analysis The overall methylation rate of the CpG island was calculated by calculating the percentage of the CpG sites in the CpG island that were methylated in the sequencing results (total methylation rate = number of methylated CpG sites / total number of CpG sites). The statistical analysis software was used to calculate the Fisher's exact probability test of the overall methylation difference of the target CpG island in the six tissues of the Fuqing goats at three stages and the methylation rate difference of each CG site, to test whether there was a methylation difference between the two groups. 9Results By determining the IMF content of the longissimus dorsi muscle of the Fuqing goats at the birth, weaning and adult stages, it was found that the IMF content of the longissimus dorsi muscle of the goats at the birth stage was 0.4% ± 0.2%, the IMF content of the longissimus dorsi muscle of the goats at the weaning stage was 0.8 ± 0.1%, and the IMF content of the longissimus dorsi muscle of the goats at the adult stage was 3.3 ± 0.3%, which was significantly higher than that at the weaning and birth stages (P < 0.01).
[0028] By analyzing the structure of the CpG island in the first intron of the ACSL5 gene of the Fuqing goats, it was found that this region contained four CpG sites (CpG1, CpG2, CpG3 and CpG4). Figure 2 To explore the overall methylation state of the CpG island in each tissue, a methylation map of the ACSL5 gene was constructed. Figure 3 Methylation detection was performed on six tissues (brain, liver, kidney, fat, longissimus dorsi muscle and biceps femoris muscle) of the goats at the birth, weaning and adult stages, 15 single clones were selected for sequencing in each tissue, and the methylation map was drawn accordingly.
[0029] The results showed that the DNA methylation level of the first intron of the ACSL5 gene in different tissues of the goats (the average value is in the brackets): Adult stage: brain tissue: 50% ~ 80% (65%); liver tissue: 53% ~ 73% (63%); kidney tissue: 75% ~ 97% (86%); fat tissue: 75% ~ 97% (86%); longissimus dorsi muscle tissue: 80% ~ 92% (86%); biceps femoris muscle tissue: 57% ~ 72% (64.5%); Weaning stage: brain tissue: 90% ~ 100% (95%); liver tissue: 42% ~ 53% (47.5%); kidney tissue: 97% ~ 100% (98.5%); fat tissue: 90% ~ 95% (92.5%); longissimus dorsi muscle tissue: 80% ~ 87% (83.5%); biceps femoris muscle tissue: 80% ~ 83% (81.5%); Newborn: brain tissue: 75%~97%(86%); liver tissue: 35%~55%(45%); kidney tissue: 88%~97%(92.5%); fat tissue: 90%~95%(92.5%); longissimus dorsi muscle tissue: 70%~85%(77.5%); gluteofemoral muscle tissue: 82%~85%(83.5%).
[0030] Further difference analysis of the DNA methylation level of the first intron of the ACSL5 gene in the six tissues of the three periods shows that: Brain tissue: from the newborn period to the adult period, the DNA methylation level of the first intron of the ACSL5 gene gradually increases, and there is a significant difference between the newborn period and the adult period (P<0.05); Liver tissue: the DNA methylation level of the first intron of the ACSL5 gene is the highest in the newborn period, and there is a significant difference between the weaning period (P<0.05); Kidney, fat and longissimus dorsi muscle tissues: from the newborn period to the adult period, the DNA methylation level of the first intron of the ACSL5 gene always maintains a high level, but there is no significant difference between the periods (P>0.05); Gluteofemoral muscle tissue: the DNA methylation level of the first intron of the ACSL5 gene is the lowest in the newborn period, and there is a significant difference between the weaning period and the adult period (P<0.05).
[0031] Pearson correlation analysis of the DNA methylation level of the first intron of the ACSL5 gene in the tissues of the three periods of Fuqing goats and the longissimus dorsi IMF content shows that the DNA methylation level of the first intron of the ACSL5 gene in the liver tissue is significantly negatively correlated with the longissimus dorsi IMF content (P=0.013).
[0032] The above results show that the DNA methylation level of the first intron of the ACSL5 gene in the liver tissue of goats can be used to assist in determining the longissimus dorsi IMF content of goats.
[0033] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method for detecting genes related to fat deposition in goats ACSL5 A primer set for detecting DNA methylation level in intron 1, characterized in that: The primer set has the following sequence: Upstream outer primer CpG1-OF: 5'-GAGTGTTTGATTTTGGAGGAAATTATT-3', Downstream outer primer CpG1-OR: 5'-CTAATCCCTTAACCTCATAAAATCCCA-3', Upstream inner primer CpG1-IF: 5'-TATTTTTTTTAGGTTTTAAGTTTTT-3', Downstream inner primer CpG1-IR: 5′-CCATCTTTAAATTCATCTCTTAACC-3′.
2. The primer set according to claim 1 is used to prepare a gene for detecting fat deposition related genes in goats ACSL5 Application of a kit for detecting DNA methylation level in intron 1.
3. A method for detecting genes related to fat deposition in goats ACSL5 A kit for detecting DNA methylation level in intron 1, characterized in that: The kit comprises the primer set according to claim 1.
4. The kit according to claim 3, wherein: The method for using the kit comprises the following steps: S1: Extract the genomic DNA of the goat to be tested and treat it with bisulfite; S2: Using bisulfite-treated genomic DNA as a template, PCR amplification was performed using the upstream outer primer CpG1-OF and the downstream outer primer CpG1-OR to obtain PCR product 1; S3: Using PCR product 1 as a template, PCR amplification was performed using the upstream inner primer CpG1-IF and the downstream inner primer CpG1-IR to obtain PCR product 2; S4: Sequence PCR product 2, analyze the sequencing results, and identify the fat deposition-related genes in the goats to be tested ACSL5 DNA methylation level of intron 1.
5. The kit according to claim 4, wherein: The genomic DNA is extracted from brain tissue, liver tissue, kidney tissue, fat tissue, longissimus dorsi muscle tissue or gluteus femoris muscle tissue of the goat to be tested.
6. The kit according to claim 4, wherein: The reaction system of the PCR amplification was as follows: 1 μL of 50 ng / μL template, 0.5 μL of 10 pmol upstream and downstream primers, 12.5 μL of 2×Premix Taq Mix, and 10.5 μL of deionized water.
7. The kit according to claim 4, wherein: The PCR amplification reaction program was as follows: 94°C for 5 min; 94°C for 30 s, 58-67°C for 50 s, 40 cycles; 72°C for 30 s; and 72°C for 5 min.
8. Use of the primer set according to claim 1 and the kit according to any one of claims 3 to 6 in preparing a product for assisting in determining the intramuscular fat content of the longissimus dorsi muscle of goats.
9. The use according to claim 8, characterized in that: According to the fat deposition related genes in goat liver tissue ACSL5 The corresponding relationship between the DNA methylation level of intron 1 and the intramuscular fat content of the longissimus dorsi muscle of goats can help determine the intramuscular fat content of the longissimus dorsi muscle of goats to be tested, and the fat deposition-related genes in goat liver tissue ACSL5 The DNA methylation level of intron 1 was significantly negatively correlated with the intramuscular fat content of the longissimus dorsi muscle of goats.