Enhancer for regulating and controlling pig ELOVL5 gene expression and identification and application of SNP (Single Nucleotide Polymorphism) of enhancer

Abstract

The invention belongs to the field of gene engineering, and discloses an enhancer for regulating and controlling pig ELOVL5 gene expression and application of the enhancer. The nucleotide sequence of the enhancer is as shown in SEQ ID No. 1 or 4. A dual luciferase report system and a CRISPR dCas9-p300 experiment prove that the enhancer can be used for remarkably enhancing the transcriptional activity of the ELOVL5 gene. Furthermore, two adjacent SNP sites exist in a region where the enhancer is combined with a transcription factor ZNF460 through identification in a pig group, and a dual-luciferase experiment shows that after the two SNP sites are mutated, the enhancer loses the expression regulation and control capability on the ELOVL5 gene. The enhancer and the SNP site thereof can be used as molecular markers for screening specific genotype individuals in pig breeding, so that the backfat thickness of a group is improved in a targeted manner, and the production performance and economic benefits of live pigs are improved.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering, and specifically relates to the identification and application of an enhancer and its SNP that regulates the expression of the porcine ELOVL5 gene. Background Art

[0002] Fat deposition is one of the core traits that influences pig production performance and economic benefits. Excessive subcutaneous fat accumulation (e.g., increased backfat thickness) significantly increases feed costs and, due to modern consumer preference for lean meat products, reduces market acceptance. From a genetic perspective, fat deposition in pigs is regulated by a multi-gene network, in which genetic variation in coding genes and their cis-regulatory elements is the primary determinant of fat deposition differences. Therefore, the precise identification of functional genes and regulatory elements associated with fat deposition has become a key target in molecular breeding for improving pork quality.

[0003] Enhancers, as non-coding DNA sequences in the genome, are an important class of cis-regulatory elements. They bind to transcription factors and cofactors to remotely regulate the transcriptional activity of target genes through spatial conformational changes such as chromatin looping. Their regulation is tissue-specific and developmental stage-specific. 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 mutation or artificial editing of enhancers has become an important strategy for improving economic traits. A typical case is the G→A mutation in intron 3 of the IGF2 gene. This mutation promotes muscle proliferation and inhibits fat deposition by enhancing the enhancer activity of the IGF2 gene, significantly increasing the lean meat percentage.

[0004] With the development of gene editing technologies such as CRISPR / Cas9, targeted modification of enhancer elements has become possible. This type of technology can fine-tune target gene expression by regulating enhancer activity without changing the coding sequence, avoiding the off-target effects and developmental toxicity that may be caused by 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 pig genome is still relatively limited. In particular, enhancers directly related to fat deposition and their key single nucleotide polymorphism (SNP) sites urgently need to be systematically explored.

[0005] Current research on the regulation of pig fat deposition primarily focuses on the functional analysis of protein-coding genes, while research on upstream non-coding regulatory elements (such as enhancers) is relatively lagging. The ELOVL5 gene, a member of the fatty acid elongase family, is involved in the synthesis of long-chain polyunsaturated fatty acids. Its expression level is closely correlated with adipocyte differentiation, but studies have yet to reveal the enhancer mechanism of its transcriptional regulation. Furthermore, although genome-wide association studies (GWAS) have mapped multiple genomic regions associated with backfat thickness, the identification of functional SNPs in these regions (particularly sites that influence enhancer binding to transcription factors) remains a lack of systematic research.

[0006] This study addresses these technical bottlenecks by identifying a functional enhancer upstream of the ELOVL5 gene through multi-omics analysis and identifying a key SNP site within this enhancer that influences the binding of the transcription factor ZNF460. This discovery provides a new target for molecular breeding of fat deposition traits in pigs, fills a gap in the understanding of 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] In response to the above-mentioned deficiencies in the existing technology, the present invention aims to provide a functional enhancer and its single nucleotide polymorphism (SNP) molecular marker related to pig fat deposition traits (especially backfat thickness), and to clarify its mechanism of action in regulating ELOVL5 gene expression, so as to provide precise genetic targets and application strategies for improving fat deposition and enhancing production performance in pig molecular breeding.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: In one embodiment, the present invention provides an enhancer for regulating 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 can enhance the transcriptional activity of the porcine ELOVL5 gene in pig cells; or the nucleotide sequence of the enhancer is a fragment of the sequence shown in SEQ ID No. 1 or 4 that is truncated to no more than 5bp, 10bp, 15bp, or 20bp, and can still enhance the transcriptional activity of the porcine ELOVL5 gene in pig cells.

[0009] In another embodiment, the present invention provides an enhancer for regulating the expression of porcine ELOVL5 gene, characterized in that the nucleotide sequence of the enhancer is shown as SEQ ID No. 1 or 4.

[0010] In another embodiment, there are two adjacent SNP sites in the enhancer of the present invention, whose genomic coordinates are calculated based on the Sscrofa11.1 genome and are located at 47089373 and 47089374 on porcine chromosome 7. The SNP sites are located in the conserved binding region of the transcription factor ZNF460.

[0011] In another embodiment, the SNP site has three genotype distributions of AG, AA and TA in the pig population, and the results of the dual luciferase experiment show that AG has an enhancer regulatory effect, while TA does not have enhancer regulatory ability.

[0012] In one embodiment, the present invention provides a method for detecting the above-mentioned SNP sites, by extracting pig genomic DNA and using PCR or sequencing to detect the genotypes of sites 47089373 and 47089374 on chromosome 7, which is used for molecular marker-assisted selection of backfat thickness traits in pig populations.

[0013] In one embodiment, the present invention provides a method for verifying the activity of the above-mentioned enhancer, using a dual-luciferase reporter system or CRISPR dCas9-p300 experiment, connecting the enhancer to the ELOVL5 gene promoter and transfecting cells to detect luciferase activity or the mRNA expression level of the ELOVL5 gene.

[0014] In one embodiment, the present invention provides the use of the aforementioned enhancer in regulating the transcriptional expression of the porcine ELOVL5 gene, which is achieved in transgenic pigs.

[0015] In one embodiment, the present invention provides an application of the above-mentioned SNP site in pig molecular breeding. By detecting the genotype of the SNP site, individuals with a specific genotype 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 pigs with other genotypes.

[0016] In one embodiment, the present invention provides a recombinant vector comprising the aforementioned enhancer sequence or the aforementioned enhancer sequence containing a SNP site, for use in a dual-luciferase reporter system or a gene editing experiment.

[0017] In one embodiment, the present invention provides a pig breeding method, which detects the genotype of a SNP site in a pig population and selects individuals with the AG genotype as breeding pigs to reduce backfat thickness and increase lean meat percentage. The genomic coordinates of the SNP site are calculated based on the Sscrofa11.1 genome and are located at 47089373 and 47089374 on pig chromosome 7. The SNP site is located in the conserved binding region of the transcription factor ZNF460.

[0018] The beneficial effects achieved by the present invention are: (1) This study used ATAC-seq, ChIP-seq, RNA-seq, and Hi-C multi-omics analysis to locate for the first time a functional enhancer (SEQ ID No. 1) 156.6 kb upstream of the ELOVL5 gene promoter in the pig genome, confirming that the enhancer remotely regulates ELOVL5 transcription through spatial chromatin interaction, thus filling the theoretical gap in the regulation of pig fat deposition in non-coding regions.

[0019] (2) The present invention discovered two adjacent SNP sites in the enhancer (47089373 and 47089374 on chromosome 7). The SNP site is a conserved region that binds to the transcription factor ZNF460. In the pig population, 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 the regulatory function of the enhancer, providing a new model for analyzing the function of SNPs in non-coding regions.

[0020] (3) The present invention can directly screen individuals with reduced backfat thickness by detecting enhancer SNP sites (AG, AA, and TA genotypes). Phenotypic measurements of the core Large White pig population showed that pigs with the AG genotype had significantly thicker backfat than those with other genotypes. This marker can be used to achieve early screening in the piglet stage through DNA testing of ear margin tissue, avoiding the long cycle and high cost of traditional phenotypic selection and significantly shortening the breeding generation interval.

[0021] (4) The present invention has a safe gene editing breeding target. By using the enhancer SNP region as the editing target and using CRISPR / Cas9 technology to modify the AA / TA→AG mutation, the enhancer activity can be precisely improved without changing the ELOVL5 coding sequence, avoiding the off-target risks and developmental toxicity that may be caused by editing the coding region. CRISPR dCas9-p300 experiments confirmed that activating this enhancer can increase ELOVL5 expression, providing a gene editing strategy for the positive regulation of fat deposition, which is suitable for the breeding of special pig breeds that require optimized fat distribution.

[0022] (5) This invention has significant cost advantages and industrial adaptability. By regulating backfat thickness and reducing fat deposition, it can reduce the carbon emission intensity of pig farming, which is consistent with the green development needs of 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Establishment of a high-fat diet-induced obese pig model and ATAC-seq sequencing; Figure 2 To identify enhancer elements that regulate ELOVL5 gene transcriptional expression for multi-omics analysis; Figure 3 This is a map of the ELOVL5 promoter and enhancer after cloning into the PGL3-Basic plasmid; Figure 4 The relative fluorescence histogram is a graph showing the activity of the enhancer verified by dual-luciferase. Figure 5 To verify the regulatory effect of enhancers on ELOVL5 gene expression using dCas9-p300 experiments; Figure 6 It is a conserved binding site for ZNF460 transcription factors in enhancers; Figure 7 Predict conserved sites for enhancer binding to ZNF460 transcription factor for Alphafold3; Figure 8 is the distribution of enhancer SNPs in pig populations; Figure 9 To verify the enhancer activity of different SNP genotypes using dual luciferase assay; Figure 10 Backfat thickness of different enhancer genotypes in the core Large White pig population. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0025] Example 1: Screening of enhancers regulating pig fat deposition 1. Experimental Materials and Treatments Experimental animals: Six healthy female Bama pigs aged 4 months with uniform body weight were randomly divided into two groups (n=3): Control group: fed with basal diet; High-fat group: A high-fat feed with lard, bile salts and cholesterol added to the basal diet; other nutritional ingredients were the same as those of the control group.

[0026] Feeding and management: free access to food and water, daily record of feed intake, slaughter after 7 months, measure back fat thickness, the results are as follows: Figure 1As shown, compared with normally fed pigs, the average weight of high-fat fed pigs increased by 4.2 kg and the average back fat increased by 0.64 cm, indicating that the induced obesity pig model was successfully constructed.

[0027] 2. Adipose Tissue Sampling and Sequencing Tissue collection: Subcutaneous fat tissue from the back was collected immediately after slaughter for DNA extraction.

[0028] ATAC-seq sequencing: Adipose tissue cell nuclei were extracted and resuspended in 100 μL Tn5 transposase reaction system (containing 2.5 μL Tn5 enzyme, Illumina), and incubated at 37°C for 30 min for transposition reaction; Equimolar sequencing adapters (Nextera XT Index Kit, Illumina) were added, and PCR amplification was performed (98°C for 30 s, 10 cycles: 98°C for 10 s, 63°C for 30 s, 72°C for 30 s, and 72°C for 5 min); The product was purified using AMPure magnetic beads, and the library quality was tested using the Qubit 4.0 fluorescence quantitative system. After quantification, 150 bp paired-end sequencing was performed on the Illumina Novaseq platform.

[0029] RNA-seq sequencing: Total RNA from adipose tissue was extracted using the RNeasy Mini Kit, and libraries were constructed using the Ribo-Zero rRNA Depletion Kit and the Illumina TruSeq strand-specific RNA library construction protocol. Illumina TruSeq Stranded mRNA library construction, Qubit dsDNA high-sensitivity kit quality control, and Novasek platform 150bp paired-end sequencing.

[0030] 3. Data Analysis ATAC-seq analysis: Data filtering: remove adapters and low-quality reads (Q < 20); The porcine genome (Sscrofa11.1) was aligned with Bowtie2, and peaks were identified using MACS2 (v2.2.7) with the following parameters: -q 0.05 --nomodel --shift -75 --extsize 150 -g 2500000000 -B --SPMR --keep-dup all; Read counts were calculated for peaks in non-promoter regions (>2 kb from TSS), and Deseq2 was used to identify differential enhancers (|log2FC|>1, P<0.05). Combined with ChIP-seq data from public databases, enhancers with significantly increased activity in the high-fat group were screened.

[0031] Through rigorous statistical tests, an enhancer (SEQID No. 1) was identified that was significantly correlated with back fat thickness after high-fat feeding. Hi-C data of porcine adipose tissue were downloaded from the NCBI public database. Through rigorous bioinformatics analysis, significant three-dimensional spatial interactions of the whole genome were identified, and the identified candidate enhancers were anchored to all potential target genes. RNA-seq data were aligned to pig transcripts using STAR software, transcriptional quantification of all protein-coding genes was performed, and Deseq2 software was used to identify genes whose expression was significantly increased after high-fat feeding. Combining differential gene expression, candidate enhancer coordinates and three-dimensional genomic interactions, the enhancer 156.6 kb upstream that regulates ELOVL5 gene expression was finally locked as the core regulatory element for candidate verification. The results of the identification 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.

[0032] Example 2: Functional verification of candidate enhancers 1. Dual-luciferase reporter assay Vector construction: The ELOVL5 promoter (SEQ ID No. 2) was amplified by PCR using DNA from Bama pig ear margin tissue as a template with 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; 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)) and inserted upstream of the promoter in the pGL3-Promoter vector to construct pGL3-Promoter-Enhancer. The constructed plasmid is described in [ 1 ]. Figure 3 .

[0033] Cell transfection and detection: The constructed vectors were transfected into HEK 293T cells. 48 hours after transfection, detection was performed using the Dual-Luciferase® Reporter Assay System (E1910, purchased from Promega) according to the manufacturer's instructions.

[0034] The 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 effect on ELOVL5 gene expression.

[0035] 2. dCas9-p300 Activation Experiment sgRNA design and vector construction: Targeting the ELOVL5 candidate enhancer sequence, six sgRNAs (sgRNA1-6) were designed using the online website https: / / www.benchling.com / . The targeting sequences were: sgRNA1: 5'-TCGGTACGATTCGAGAGGGA-3' (SEQ ID No.10) sgRNA2: 5'-GTACGATTCGAGAGGGACGG-3' (SEQ ID No.11) sgRNA3: 5'-ACCCAAGTGCGCAGACACAG-3' (SEQ ID No.12) sgRNA4: 5'-GGGCTGCCTGAACCTAGTGG-3' (SEQ ID No.13) sgRNA5: 5'-GCAGACGTCCCCAGACACAA-3' (SEQ ID No.14) sgRNA6: 5'-CCATCGGTATGGCAGTCCAA-3' (SEQ ID No. 15).

[0036] The above six sgRNAs were cloned into the pGL-U6-sgRNA-tdTomato vector and co-transfected with the pCDNA-dCas9-p300core vector into Bama pig ear fibroblasts (PEF).

[0037] RNA extraction and QPCR: 48 h after transfection, RNA was extracted using the TRIzol method. 200 μL of chloroform was added to the TRIzol, vortexed for 15 seconds, allowed to stand for 10 minutes, and then centrifuged at 12,000 rpm at 4°C for 10 minutes. The upper aqueous phase was transferred to a new 1.5 mL centrifuge tube, and an equal volume of pre-cooled isopropanol was added. The tube was mixed by inversion and allowed to stand for 10 minutes. The tube was centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was carefully aspirated and a white precipitate was visible at the bottom of the tube. 1 ml of pre-cooled 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 at 4°C for 5 minutes, and washed twice. Use a pipette to remove as much residual ethanol as possible from the centrifuge tube. Open the tube and air-dry it at room temperature for 5 minutes. Add an appropriate amount of enzyme-free sterile water to 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). QPCR was then used to detect the mRNA level of the downstream gene ELOVL5.

[0038] The results showed that among the six sgRNAs designed for the ELOVL5 enhancer sequence, five vectors with corresponding sequences (sgRNA 1 / 2 / 3 / 5 / 6) significantly increased the expression level of the ELOVL5 gene to an average of approximately 1.5 times, further demonstrating the regulatory effect of the enhancer on the expression of the ELOVL5 gene (Figure 5).

[0039] Example 3: Functional verification of enhancer SNP sites 1. Transcription factor binding site prediction Download the transcription factor motif data from the JASPAR database and use the MEME online website to predict the transcription factor binding sites in the ELOVL5 enhancer sequence. An extremely significant transcription factor binding site, ZNF460, was identified at the center of the enhancer. The results are as follows: Figure 6 The conserved sites of enhancer binding to ZNF460 were predicted using the Alphafold3 online website. The results are shown in Figure 7 shown.

[0040] 2. Enhancer SNP Detection 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 calling, two adjacent SNPs were identified at positions 13 and 14 of the ZNF460 transcription factor binding site. Their genomic coordinates are chromosome 7: 47089373 and 47089374. The genotype analysis of the SNPs in the population is as follows. Figure 8 As shown, the SNP site has three genotype distributions in the pig population: AG, AA and TA.

[0041] 3. Dual luciferase assay to verify the regulatory role of enhancer SNP Based on the information of these two adjacent SNPs, three enhancer fragments with different genotypes (AG, AA, TA, as shown in SEQ ID No. 3, 4, and 5) were designed and synthesized in vitro. The synthesized enhancer sequences were transferred into the pGL3-Promoter vector. The constructed vectors were transfected into porcine SVF cells. 48 hours after transfection, the Dual-Luciferase® Reporter Assay System (E1910, purchased from Promega) kit was used for detection according to its instructions. The experimental results are shown in Figure 2. Figure 9 As shown, changing one of the SNPs 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 effect, thus proving the regulatory effect of these two adjacent SNP sites on ELOVL5 gene expression.

[0042] 4. Production indicators of different enhancer genotypes in the core Large White pig population A core group of 48 Large White pigs obtained from a partner was used to identify the backfat thickness of each pig through precise measurement. The genotype of enhancer SNPs was identified by extracting DNA from the ear tissue of each pig to evaluate the association between each genotype and production phenotype. Because the AG genotype mainly appears in Duroc pigs (results see Figure 8 ), so the detection rate of AG genotype in Large White pigs is low, such as Figure 10 As shown in the figure, the average backfat thickness of the AG genotype is only 0.78 cm, significantly lower than the 1.16 cm of the AA genotype and the 1.36 cm of the TA genotype. This indicates that the AG genotype is more closely associated with the low backfat trait and has important application value in breeding.

[0043] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An enhancer for regulating porcine ELOVL5 gene expression, characterized by: The nucleotide sequence of the enhancer is shown in SEQ ID No. 1 or 4.

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

3. A method for verifying the enhancer activity of claim 1, characterized in that: The enhancer is connected to the ELOVL5 gene promoter using a dual luciferase reporter system or a dCas9-p300 assay and then transfected into cells to detect luciferase activity or the mRNA expression level of the ELOVL5 gene.

4. Use of the enhancer according to claim 1 in regulating the transcriptional expression of the porcine ELOVL5 gene, characterized in that: This is achieved using genetically modified pigs.

5. Application of a SNP site in pig molecular breeding, characterized by: By detecting the genotype of the SNP site, individuals with a specific genotype are screened for breeding, thereby improving the backfat thickness of the pig population, wherein the backfat thickness of pigs with the AG genotype is significantly greater than that of the AA and TA genotypes; The genomic coordinates of the SNP site are calculated based on the Sscrofa11.1 genome, located at 47089373 and 47089374 of pig chromosome 7, and there are three genotypes: AG, AA and TA.

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

7. The recombinant vector according to claim 1, characterized in that It is used in dual-luciferase reporter systems or gene editing experiments.

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

Images