Identification and application of an enhancer and its SNP regulating porcine ELOVL5 gene expression

Abstract

This invention belongs to the field of genetic engineering and discloses an enhancer that regulates the expression of the porcine ELOVL5 gene and its applications. The nucleotide sequence of the enhancer is shown in SEQ ID No. 1 or 4. Experiments using a dual-luciferase reporter system and CRISPR dCas9-p300 confirmed that this enhancer significantly enhances the transcriptional activity of the ELOVL5 gene. Further analysis in a pig population revealed two adjacent SNP sites in the region where the enhancer binds to the transcription factor ZNF460. Dual-luciferase experiments showed that mutations at these two SNP sites resulted in the enhancer losing its ability to regulate ELOVL5 gene expression. This enhancer and its SNP sites can serve as molecular markers for screening individuals with specific genotypes in pig breeding, specifically improving backfat thickness, and enhancing pig production performance and economic benefits.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering, specifically relating to the identification and application of an enhancer that regulates the expression of the porcine ELOVL5 gene and its SNPs. Background Technology

[0002] Fat deposition is one of the core traits affecting the production performance and economic benefits of pigs. Excessive subcutaneous fat accumulation (such as increased backfat thickness) significantly increases feed costs, and the preference of modern consumers for lean meat products leads to decreased market acceptance. From a genetic perspective, fat deposition in pigs is regulated by a multi-gene network, with genetic variations in coding genes and their cis-regulatory elements being the main factors determining differences in fat deposition. Therefore, accurately identifying functional genes and regulatory elements related to fat deposition has become a key target for improving pork quality in the field of molecular breeding.

[0003] Enhancers, as non-coding DNA sequences in the genome, are important cis-regulatory elements. They regulate the transcriptional activity of target genes over long distances through spatial conformational changes such as chromatin circularization, by binding to transcription factors and cofactors. Their regulation exhibits tissue specificity and developmental stage specificity. For example, in adipose tissue, specific enhancers can promote adipocyte differentiation and lipid accumulation by activating the expression of lipid metabolism-related genes (such as PPARγ and FABP4). In pig genetic breeding, natural variations or artificial editing of enhancers have become important strategies for improving economically important traits. A typical example is the G→A mutation in intron 3 of the IGF2 gene. This mutation enhances the enhancer activity of the IGF2 gene, promoting muscle proliferation and inhibiting fat deposition, thus significantly increasing lean meat percentage.

[0004] With the development of gene editing technologies such as CRISPR / Cas9, targeted modification of enhancer elements has become possible. These technologies can finely regulate target gene expression by modulating enhancer activity without altering the coding sequence, avoiding the off-target effects and developmental toxicity that can occur with traditional gene knockout. For example, activating muscle-specific enhancers can promote muscle fiber growth, while inhibiting adipose tissue enhancers can reduce lipid deposition. However, the number of functional enhancers identified in the porcine genome remains relatively limited, especially enhancers directly related to lipid deposition and their key single nucleotide polymorphism (SNP) sites, which urgently require systematic exploration.

[0005] Current research on the regulation of fat deposition in pigs mainly focuses on the functional analysis of protein-coding genes, while research on upstream non-coding regulatory elements (such as enhancers) lags behind. The ELOVL5 gene, as a member of the fatty acid elongase family, participates in the synthesis of long-chain polyunsaturated fatty acids, and its expression level is closely related to adipocyte differentiation; however, no studies have yet revealed its transcriptional regulatory enhancer mechanism. Furthermore, although genome-wide association studies (GWAS) have located several genomic regions related to backfat thickness, the identification of functional SNPs (especially sites affecting enhancer binding to transcription factors) in these regions still lacks systematic research.

[0006] Based on the aforementioned technical bottlenecks, this invention screened for a functional enhancer upstream of the ELOVL5 gene through multi-omics analysis and identified a key SNP site within this enhancer that affects the binding of the transcription factor ZNF460. This discovery provides a new target for molecular breeding of the pig fat deposition trait, fills a gap in the transcriptional regulation mechanism of the ELOVL5 gene, and provides technical support for improving backfat thickness through enhancer SNP marker-assisted selection. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention aims to provide a functional enhancer and its single nucleotide polymorphism (SNP) molecular marker related to the fat deposition trait in pigs (especially backfat thickness), and to elucidate its mechanism of action in regulating ELOVL5 gene expression, providing precise genetic targets and application strategies for improving fat deposition and enhancing production performance in pig molecular breeding.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In one embodiment, the present invention provides an enhancer that regulates the expression of the porcine ELOVL5 gene, wherein the nucleotide sequence of the enhancer has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the sequence shown in SEQ ID No. 1 or 4, and is capable of enhancing the transcriptional activity of the porcine ELOVL5 gene in porcine cells; or the nucleotide sequence of the enhancer is a fragment of the sequence shown in SEQ ID No. 1 or 4 truncated to no more than 5bp, 10bp, 15bp, or 20bp, and is still capable of enhancing the transcriptional activity of the porcine ELOVL5 gene in porcine cells.

[0010] In another embodiment, the present invention provides an enhancer that regulates the expression of the porcine ELOVL5 gene, characterized in that the nucleotide sequence of the enhancer is as shown in SEQ ID No. 1 or 4.

[0011] In another embodiment, the enhancer of the present invention contains two adjacent SNP sites with genomic coordinates calculated using the Sscrofa11.1 genome, located on pig chromosome 7 at coordinates 47089373 and 47089374. These SNP sites are located in the conserved binding region of transcription factor ZNF460.

[0012] In another embodiment, the SNP site is distributed in three genotypes in the pig population: AG, AA, and TA. The results of the dual-luciferase assay show that AG has an enhancer regulatory role, while TA does not have an enhancer regulatory ability.

[0013] In one embodiment, the present invention provides a method for detecting the above-mentioned SNP sites, which involves extracting pig genomic DNA and using PCR or sequencing to detect the genotypes of sites 47089373 and 47089374 on chromosome 7, for molecular marker-assisted selection of backfat thickness traits in pig populations.

[0014] In one embodiment, the present invention provides a method for verifying the activity of the above-mentioned enhancer, which involves using a dual-luciferase reporter system or a CRISPR dCas9-p300 assay to link the enhancer to the ELOVL5 gene promoter and then transfecting cells to detect luciferase activity or the mRNA expression level of the ELOVL5 gene.

[0015] In one embodiment, the present invention provides the application of the above-mentioned enhancer in regulating the transcriptional expression of the porcine ELOVL5 gene, which is implemented in transgenic pigs.

[0016] In one embodiment, the present invention provides the application of the above-mentioned SNP loci in pig molecular breeding. By detecting the genotype of the SNP loci, individuals with specific genotypes are screened for breeding to improve the backfat thickness of the pig population. The backfat thickness of pigs with the AG genotype is significantly greater than that of other genotypes.

[0017] In one embodiment, the present invention provides a recombinant vector comprising the above-described enhancer sequence or the above-described enhancer sequence containing SNP sites, for use in a dual-luciferase reporter system or gene editing experiments.

[0018] In one embodiment, the present invention provides a pig breeding method that selects individuals with the AG genotype as breeding pigs by detecting the SNP locus genotype in a pig population in order to reduce backfat thickness and increase lean meat percentage. The genomic coordinates of the SNP locus are calculated based on the Sscrofa11.1 genome and are located on chromosome 7 of pig at 47089373 and 47089374. The SNP locus is located in the conserved binding region of transcription factor ZNF460.

[0019] The beneficial effects achieved by this invention are as follows:

[0020] (1) Through multi-omics analysis using ATAC-seq, ChIP-seq, RNA-seq and Hi-C, this invention has for the first time located a functional enhancer (SEQ ID No. 1) 156.6 kb upstream of the ELOVL5 gene promoter in the pig genome. This confirms that the enhancer regulates ELOVL5 transcription over long distances through chromatin spatial interaction, filling the theoretical gap in the regulation of pig fat deposition in non-coding regions.

[0021] (2) This invention discovered two adjacent SNP sites (chromosome 7, 47089373 and 47089374) in the enhancer. These SNP sites are conserved regions that bind to the transcription factor ZNF460. In pig populations, there are three different genotypes: AG, AA, and TA. AG and AA can bind to ZNF460 and thus regulate the expression of the ELOVL5 gene, while TA loses its enhancer regulatory function. This provides a new model for analyzing the function of non-coding region SNPs.

[0022] (3) This invention can directly screen individuals with reduced backfat thickness by detecting enhancer SNP sites (AG, AA, and TA genotypes). Phenotypic analysis results of the core Large White pig population show that pigs with the AG genotype have significantly thicker backfat than those with other genotypes. This marker can be used for early screening in piglets through ear tissue DNA detection, avoiding the long cycle and high cost of traditional phenotypic breeding, and significantly shortening the generation interval of breeding.

[0023] (4) This invention provides a safe gene-editing breeding target. By using the enhancer SNP region as the editing target and employing CRISPR / Cas9 technology to directionally modify the AA / TA→AG mutation, the enhancer activity can be precisely increased without altering the ELOVL5 coding sequence, avoiding off-target risks and developmental toxicity that may be caused by coding region editing. CRISPR dCas9-p300 experiments have confirmed that activating this enhancer can upregulate ELOVL5 expression, providing a gene-editing strategy for the positive regulation of fat deposition, and is suitable for the breeding of specialty pig breeds that require optimization of fat distribution.

[0024] (5) This invention has significant cost advantages and industrial adaptability. By controlling the backfat thickness to reduce fat deposition, it can reduce the carbon emission intensity of pig farming, which meets the needs of green development in animal husbandry. It provides molecular markers with independent intellectual property rights for the breeding of high-quality lean pig breeds, reduces dependence on foreign breeding technologies, and enhances the core competitiveness of my country's pig industry. Attached Figure Description

[0025] Figure 1 Constructing an obese pig model induced by a high-fat diet and performing ATAC-seq sequencing;

[0026] Figure 2 To identify enhancer elements regulating the transcriptional expression of the ELOVL5 gene in a multi-omics analysis;

[0027] Figure 3 The map shows the results of cloning the ELOVL5 promoter and enhancer into the PGL3-Basic plasmid;

[0028] Figure 4 A relative fluorescence bar chart for verifying enhancer activity using dual-luciferase assay;

[0029] Figure 5 To verify the regulatory role of enhancers on ELOVL5 gene expression using dCas9-p300 experiments;

[0030] Figure 6 This is a conserved binding site for the ZNF460 transcription factor in the enhancer;

[0031] Figure 7 Conserved sites for Alphafold3 enhancer binding to ZNF460 transcription factor;

[0032] Figure 8 To enhance the distribution of sub-SNPs in the pig population;

[0033] Figure 9 To verify the enhancer activity of different SNP genotypes using a dual-luciferase assay;

[0034] Figure 10 The backfat thickness of different enhancer genotypes in the core Large White pig population. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0036] Example 1: Screening of enhancers related to the regulation of fat deposition in pigs

[0037] 1. Experimental Materials and Processing

[0038] Experimental animals: Six healthy female Bama pigs aged 4 months with uniform weight were selected and randomly divided into two groups (n=3):

[0039] Control group: fed with a basal diet;

[0040] High-fat group: The basal diet was supplemented with a high-fat diet containing lard, bile salts and cholesterol, while other nutrients were the same as the control group.

[0041] Feeding and management: Free access to feed and water; daily feed intake recorded; slaughtered after 7 months; backfat thickness measured; results as follows. Figure 1 As shown, compared with normally fed pigs, high-fat fed pigs had an average weight gain of 4.2 kg and an average backfat gain of 0.64 cm, indicating that the induced obesity pig model was successfully constructed.

[0042] 2. Adipose tissue sampling and sequencing

[0043] Tissue collection: Subcutaneous adipose tissue was collected rapidly after slaughter to extract DNA.

[0044] ATAC-seq sequencing:

[0045] Nuclei were extracted from adipose tissue cells and resuspended in 100 μL of Tn5 transposase reaction system (containing 2.5 μL Tn5 enzyme, Illumina), and incubated at 37℃ for 30 min to carry out the transposase reaction;

[0046] Add equimolar sequencing adapters (Nextera XT Index Kit, Illumina), and perform PCR amplification (98℃ 30 s, 10 cycles: 98℃ 10 s, 63℃ 30 s, 72℃ 30 s, 72℃ 5 min).

[0047] The purified products from AMPure magnetic beads were analyzed for library quality using the Qubit 4.0 quantitative PCR system. After quantification, 150 bp paired-end sequencing was performed on the Illumina Novaseq platform.

[0048] RNA-seq sequencing:

[0049] Total RNA was extracted from adipose tissue using the RNeasy Mini Kit, and libraries were constructed sequentially using the Ribo-Zero rRNA removal kit and the Illumina TruSeq strand-specific RNA library construction protocol.

[0050] Library preparation using Illumina TruSeq Stranded mRNA, quality control using the Qubit dsDNA high-sensitivity kit, and 150bp paired-end sequencing using the Novaseq platform.

[0051] 3. Data Analysis

[0052] ATAC-seq analysis:

[0053] Data filtering: Remove connectors and low-quality reads (Q<20);

[0054] Bowtie2 alignment of the pig genome (Sscrofa11.1), MACS2 (v2.2.7) peak identification, parameters: -q 0.05 --nomodel --shift -75 --extsize 150 -g 2500000000 -B --SPMR --keep-dup all;

[0055] Read counts were performed on the peak in the non-promoter region (distance from TSS > 2 kb), and differential enhancers were identified by Deseq2 (|log2FC|>1, P<0.05). Combined with ChIP-seq data from public databases, enhancers with significantly increased activity in the high-lipid group were screened.

[0056] Through rigorous statistical testing, an enhancer (SEQ ID No. 1) significantly correlated with backfat thickness after high-fat feeding was identified. Hi-C data of porcine adipose tissue were downloaded from the NCBI public database, and significant three-dimensional interactions across the entire genome were identified through rigorous bioinformatics analysis. The identified candidate enhancer was then anchored to all potential target genes. RNA-seq data were aligned to porcine transcripts using STAR software, and transcriptional quantification of all protein-coding genes was performed. Deseq2 software was used to identify genes whose expression significantly increased after high-fat feeding. Combining differential gene expression binding, candidate enhancer coordinates, and three-dimensional genome interactions, an enhancer located 156.6 kb upstream of the ELOVL5 gene expression was ultimately identified as the core regulatory element for candidate validation. The identification results are as follows: Figure 2 As shown, this enhancer can specifically target and regulate the expression of the ELOVL5 gene through chromatin folding, promoting its high expression in the high-fat diet group.

[0057] Example 2: Functional Verification of Candidate Enhancers

[0058] 1. Dual-luciferase reporter assay

[0059] Carrier construction:

[0060] Using DNA from the ear margin tissue of Bama pigs as a template, the ELOVL5 promoter (SEQ ID No. 2) was amplified by PCR using primers PE-F / PE-R (5'-GTGGGAAACCCAAGGATTGA-3' (SEQ ID No. 6) / 5'-CTCCTGGGCGTGGGGAGAGC-3' (SEQ ID No. 7)), and cloned into the pGL3-Basic vector, named pGL3-Promoter;

[0061] The candidate enhancer sequence (SEQ ID No. 1) was amplified using primers EE-F / EE-R (5'-CTCCCCAATCCCAGCACTCC-3' (SEQ ID No. 8) / 5'-GTGGGTGTGTCCGGCCCTTT-3' (SEQ ID No. 9)). This sequence was then inserted upstream of the promoter in the pGL3-Promoter vector to construct pGL3-Promoter-Enhancer. The constructed plasmid is described in [link to plasmid description]. Figure 3 .

[0062] Cell transfection and detection:

[0063] The constructed vectors were transfected into HEK 293T cells. Forty-eight hours after transfection, the samples were analyzed using the Dual-Luciferase® Reporter Assay System (E1910, Promega) kit, following the manufacturer's instructions.

[0064] Experimental results are as follows Figure 4 As shown, compared with the pGL3-Promoter group, the fluorescence value of the pGL3-Promoter-Enhancer group was significantly increased by 2.13 times, indicating that the vector corresponding to the enhancer sequence showed enhancer activity, thus proving its regulatory role in ELOVL5 gene expression.

[0065] 2. dCas9-p300 activation experiment

[0066] sgRNA design and vector construction:

[0067] Six sgRNAs (sgRNA1-6) were designed using the online website https: / / www.benchling.com / to target candidate enhancer sequences of ELOVL5. The target sequences are as follows:

[0068] sgRNA1: 5'-TCGGTACGATTCGAGAGGGA-3' (SEQ ID No.10)

[0069] sgRNA2: 5'-GTACGATTCGAGAGGGACGG-3' (SEQ ID No.11)

[0070] sgRNA3: 5'-ACCCAAGTGCGCAGACACAG-3' (SEQ ID No.12)

[0071] sgRNA4: 5'-GGGCTGCCTGAACCTAGTGG-3' (SEQ ID No.13)

[0072] sgRNA5: 5'-GCAGACGTCCCCAGACACAA-3' (SEQ ID No.14)

[0073] sgRNA6: 5'-CCATCGGTATGGCAGTCCAA-3' (SEQ ID No. 15).

[0074] The above 6 sgRNAs were cloned into the pGL-U6-sgRNA-tdTomato vector and co-transfected with the pCDNA-dCas9-p300core vector into Bama pig peripheral fibroblasts (PEF).

[0075] RNA extraction and qPCR:

[0076] 48 h after transfection, RNA was extracted using the TRIzol method. 200 μL of chloroform was added to the TRIzol solution and vortexed for 15 s. After standing for 10 min, the solution was centrifuged at 12,000 rpm and 4 °C for 10 min. The supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of pre-chilled isopropanol was added. The mixture was inverted and allowed to stand for 10 min. After centrifugation at 12,000 rpm and 4 °C for 10 min, the supernatant was carefully aspirated. A white precipitate was visible at the bottom of the tube. 1 mL of pre-chilled 75% ethanol (prepared with enzyme-free sterile water) was added to each tube to wash the precipitate. The tube was centrifuged at 12,000 rpm and 4 °C for 5 min, and washed twice. Use a pipette tip to remove as much residual ethanol as possible from the centrifuge tube. Open the centrifuge tube and allow it to air dry at room temperature for 5 minutes. Add an appropriate amount of enzyme-free sterile water and dissolve the precipitate on ice. After measuring the concentration, prepare cDNA according to the instructions of the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit (RR047A, purchased from Takara). Further, detect the mRNA level of the downstream gene ELOVL5 by qPCR.

[0077] The results showed that among the six sgRNAs designed targeting the ELOVL5 enhancer sequence, five vectors (sgRNA 1 / 2 / 3 / 5 / 6) significantly increased the expression level of the ELOVL5 gene by an average of about 1.5 times, thus further demonstrating the regulatory role of enhancers on ELOVL5 gene expression (Figure 5).

[0078] Example 3: Functional verification of enhancer SNP sites

[0079] 1. Prediction of transcription factor binding sites

[0080] Transcription factor motif data were downloaded from the JASPAR database. The MEME online website was used to predict transcription factor binding sites in the ELOVL5 enhancer sequence. A highly significant transcription factor binding site, ZNF460, was identified at the center of the enhancer. The results are as follows: Figure 6 As shown. The conserved binding sites of enhancers to ZNF460 were predicted using the Alphafold3 online website, and the results are as follows. Figure 7 As shown.

[0081] 2. Enhancer SNP detection

[0082] Whole-genome resequencing data (average depth 30×) of 734 pigs (including Large White, Landrace, and Meishan pigs) and 7 wild boars were downloaded from NCBI. After sequence alignment and SNP call analysis, two adjacent SNP sites were identified at positions 13 and 14 of the ZNF460 transcription factor binding site, with genomic coordinates of chromosome 7: 47089373 and 47089374. Genotypic analysis of the SNPs in the population is as follows: Figure 8 As shown, the SNP loci exhibit three genotypes in the pig population: AG, AA, and TA.

[0083] 3. Dual-luciferase assay to verify the regulatory role of enhancer SNPs.

[0084] Based on the information from these two adjacent SNPs, enhancer fragments for three different genotypes (AG, AA, and TA, as shown in SEQ ID No. 3, 4, and 5) were designed and synthesized in vitro. The synthesized enhancer sequences were transformed into the pGL3-Promoter vector. The constructed vectors were then transfected into porcine SVF cells. Forty-eight hours after transfection, the results were analyzed using the Dual-Luciferase® ReporterAssay System (E1910, purchased from Promega) kit, following the manufacturer's instructions. The experimental results are as follows: Figure 9 As shown, changing one SNP has little effect on the regulatory activity of the enhancer, but changing both SNPs at the same time will cause the enhancer to lose its regulatory function, thus proving the regulatory role of these two adjacent SNP sites on ELOVL5 gene expression.

[0085] 4. Production indicators of different enhancer genotypes in the core Large White pig population

[0086] Forty-eight core Large White pigs were obtained from our partner, and the backfat thickness of each pig was determined using precise measurement methods. Ear tissue DNA was extracted from each pig to identify the genotype of enhancer SNPs, in order to assess the association between each genotype and the production phenotype. Since the AG genotype is mainly found in Duroc pigs (see results...),... Figure 8 Therefore, the detection rate of the AG genotype in Large White pigs is low, such as Figure 10 As shown, the average backfat thickness of the AG genotype is only 0.78 cm, significantly lower than that of the AA genotype (1.16 cm) and the TA genotype (1.36 cm). This indicates that the AG genotype is more closely associated with the low backfat trait and has important application value in breeding.

[0087] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. An enhancer that regulates the expression of the porcine ELOVL5 gene, characterized in that: The nucleotide sequence of the enhancer is shown in SEQ ID No.

1.

2. A method for detecting SNP sites, characterized in that: By extracting pig genomic DNA, the genotype of the SNP loci is detected by PCR or sequencing for molecular marker-assisted selection of backfat thickness traits in pig populations. The genomic coordinates of the SNP loci, calculated using the Sscrofa11.1 genome, are located on chromosome 7 of pigs at 47089373 and 47089374, and there are three genotypes: AG, AA, and TA.

3. A method for verifying the activity of the enhancer according to claim 1, characterized in that: Using a dual-luciferase reporter system or a dCas9-p300 assay, the enhancer was linked to the ELOVL5 gene promoter and transfected into cells. Luciferase activity or the mRNA expression level of the ELOVL5 gene was then detected.

4. The application of the enhancer described in claim 1 in regulating the transcriptional expression of the porcine ELOVL5 gene, characterized in that, It is used to achieve this in genetically modified pigs.

5. An application of a SNP site in pig molecular breeding, characterized in that: By detecting the genotype of the SNP locus, individuals with specific genotypes are selected for breeding to improve the backfat thickness of the pig population. Among them, the backfat thickness of pigs with the AG genotype is significantly greater than that of pigs with the AA and TA genotypes. The genomic coordinates of the SNP loci, calculated using the Sscrofa11.1 genome, are located on chromosome 7 of pigs at 47089373 and 47089374, and there are three genotypes: AG, AA, and TA.

6. A recombinant vector, characterized in that: The vector comprises the enhancer sequence as described in claim 1.

7. The recombinant vector as described in claim 6, characterized in that, It is used in dual-luciferase reporter systems or gene editing experiments.

8. A method for breeding pigs, characterized in that: By detecting the SNP locus genotype in the pig population, individuals with the AG genotype were selected as breeding pigs to reduce backfat thickness and increase lean meat percentage; The SNP site's genomic coordinates, calculated using the Sscrofa11.1 genome, are located on pig chromosome 7 at coordinates 47089373 and 47089374. The SNP site is located in the conserved binding region of transcription factor ZNF460.

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