Pig acsl3 gene promoter region snp marker and its application in pig breeding against prrsv

Abstract

The application provides a SNP marker in a pig ACSL3 gene promoter region and application thereof in pig PRRS resistance breeding. SNPs in the ACSL3 gene promoter region are analyzed by using whole genome resequencing data, it is found that three single nucleotide variations in the ACSL3 gene promoter are related to pig PRRS resistance, and it is found that a CCA haplotype can improve the PRRS resistance of a population, and thus early selection of individuals can be implemented by using the molecular marker, and pig PRRS resistant varieties can be selected. The application provides a reliable molecular marker for genetic improvement of pig PRRS resistance, and has important significance for pig disease resistance breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetics, and in particular to a porcine ACSL3 gene promoter region SNP marker and application thereof in porcine PRRSV-resistant breeding. Background Art

[0002] Blue ear disease, also known as porcine reproductive and respiratory syndrome (PRRS), is a viral infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV), characterized by respiratory disorders and reproductive failure in pigs of all ages. The PRRSV genome is highly variable and prone to recombination, making vaccines ineffective in suppressing its spread. Breeding for disease resistance enhances host resistance to PRRSV at the genetic level, fundamentally curbing its prevalence.

[0003] Fatty acids play a vital role in physiological processes, participating in the formation of numerous lipids, including triacylglycerols and cholesterol esters, the formation of structural lipids such as phospholipids, conversion to alcohols or aldehydes, carbon addition and reduction, double bond insertion and removal, and covalent binding to proteins (Kuwata H and Hara S 2019, Quan J et al 2021, Soupene E and Kuypers FA 2008). Before entering these metabolic processes, free fatty acids must be converted to activated fatty acids by forming thioester bonds with coenzyme A, catalyzed by members of the acyl-CoA synthetases (ACSs) family. With the completion of genome sequencing across multiple species, 26 confirmed ACS family members have been identified using highly conserved amino acid sequence motifs (Watkins PA et al 2007). Among them, long-chain acyl-CoA synthetase (ACSL) within the acyl-CoA synthetase family is a key enzyme in the synthesis of long-chain fatty acids. The ACSL family typically consists of five members: ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6. While each member is distributed differently within the body, they all function to activate long-chain fatty acids. ACSL3, also known as ACS3, is primarily located in the periphery of adipogenic cells and within the endoplasmic reticulum (Kassan A et al 2013, Poppelreuther M et al 2012). ACSL3 has high substrate specificity for lauric acid, myristic acid, and eicosapentaenoic acid ethyl ester, preferentially activating both saturated and unsaturated fatty acids (Fujino et al 1996).

[0004] As research on ACSL3 deepens, its biological roles are being revealed to be extensive. Li et al. found that ACSL3 plays an important role as a negative regulator of ferroptosis after brain injury through the GPX4-ACSL3 axis (Li M et al 2022). Using targeted siRNA to interfere with ACSL3 expression in Huh7 cells inhibited the secretion of apolipoprotein B, a major component of lipoproteins, leading to rapid lipoprotein degradation and the secretion of HCV virions in host cells (Nchoutmboube JA et al 2013). Yao et al. found that poliovirus replication efficiency was significantly downregulated after treating cells with siRNA targeting ACSL3 (Yao H and Ye J 2008). Furthermore, ACSL3 has relatively complex functions at different stages of development in different types of cancer, such as participating in lipid synthesis and deposition in the early stages of carcinogenesis and increasing lipid utilization in late-stage breast and prostate cancer (Tang Y et al 2018, Wright et al 2017).

[0005] Currently, there are no reports of SNP markers in the promoter region of the porcine ACSL3 gene mediating resistance to PRRSV. Therefore, studying SNPs in the ACSL3 gene and identifying their relationship with PRRSV resistance would provide a theoretical basis for molecular marker-assisted breeding, allowing for the rapid and convenient selection of PRRSV-resistant individuals. Summary of the Invention

[0006] The present invention selects ACSL3 as a candidate gene for resistance to blue ear disease by comparing the genetic and gene expression differences between disease-resistant individuals and susceptible individuals in an artificially infected PRRSV resource group. By searching for genetic variations in the promoter region of the porcine ACSL3 gene, molecular markers related to porcine blue ear disease resistance and their detection methods are explored, and applied to pig genetic breeding to establish a pig molecular marker-assisted breeding method, providing genetic markers and technical methods for the selection and breeding of porcine blue ear disease-resistant individuals.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, a molecular marker related to blue ear disease resistance in the ACSL3 gene is provided, and its nucleotide sequence is SEQ ID NO.1. The molecular marker is obtained by resequencing the whole genome of a PRRSV artificially infected resource population to obtain sequencing data, extracting all SNPs in the ACSL3 gene promoter region, and determining that three highly linked SNP sites in the porcine ACSL3 gene promoter region, namely 15:124741207, 15:124741301 and 15:124741312 (the porcine reference genome is Sscrofa11.1), may be associated with blue ear disease resistance. It should be understood by those skilled in the art that in the present invention, any fragment including one of the 1223rd base, the 1317th base or the 1328th base of the sequence shown in SEQ ID NO.1 can be used as a molecular marker, preferably a fragment including these three sites. Therefore, the molecular marker of the present invention is a fragment of the nucleotide sequence shown in SEQ ID NO.1, which includes at least one of the 1223rd base, the 1317th base or the 1328th base of the sequence shown in SEQ ID NO.1, and preferably includes a fragment of the three sites of the 1223rd base, the 1317th base and the 1328th base.

[0009] Furthermore, the present invention combined frequency distribution and linkage disequilibrium analysis to identify three variant sites located in the ACSL3 promoter region as candidate sites: 15:124741207, 15:124741301, and 15:124741312. The variant types of the three candidate sites were identified as follows: the SNP site 124741207 on chromosome 15 was a G>C mutation, the SNP site 124741301 was a C>T mutation, and the SNP site 124741312 was an A>C mutation.

[0010] Furthermore, based on the fact that the three variant sites 15:124741207, 15:124741301 and 15:124741312 are strongly linked, dual-luciferase vectors were constructed for the dominant haplotypes CCA and GTC of the corresponding three SNP sites, and expression experiments were carried out using different cells. The results showed that the dual-luciferase activity of the CCA haplotype was significantly reduced.

[0011] Finally, based on the regulation of ACSL3 gene promoter activity by CCA haplotype and GTC haplotype, the CCA haplotype caused a decrease in ACSL3 gene expression. siRNA was designed targeting different interference targets of the ACSL3 gene. The results showed that interfering with the expression of the ACSL3 gene could inhibit PRRSV proliferation.

[0012] Based on this, the present invention provides a molecular marker method, wherein the molecular marker is located at the SNP site 124741207 on chromosome 15 of the pig reference genome, which is a G>C mutation, the SNP site 124741301 is a C>T mutation, and the SNP site 124741312 is an A>C mutation. These three sites are detected, and the CCA haplotype of these three sites is the dominant haplotype.

[0013] In a second aspect, the present invention provides a use of the SNP molecular marker described in the first aspect in pig genetic breeding for resistance to blue ear disease. The SNP site is located at position 124741312 on chromosome 15 of the pig reference genome; the pig reference genome is Sscrofa11.1, and the SNP site at position 124741312 is an A>C mutation.

[0014] In a third aspect, the present invention provides a method for detecting a SNP molecular marker as described in the first aspect, comprising the following steps:

[0015] Using the pig genomic DNA to be tested as a template, the ACSL3 gene promoter region fragment is amplified by PCR, and then the genotype of the SNP site located in the ACSL3 gene promoter region is identified, wherein the SNP site is one or more of the SNP sites at positions 124741207, 124741301, and 124741312 on chromosome 15 of the pig reference genome; the pig reference genome is Sscrofa11.1, the SNP site at position 124741207 is a G>C mutation, the SNP site at position 124741301 is a C>T mutation, and the SNP site at position 124741312 is an A>C mutation (that is, the SNP site is one or more of 15:124741207G>C, 15:124741301C>T, and 15:124741312A>C).

[0016] Preferably, the PCR amplification primer design template can refer to the following region: 15:124739985-15:124741985.

[0017] In a fourth aspect, the present invention provides a primer for specifically amplifying the SNP site related to porcine PRRSV resistance in the promoter region of the porcine ACSL3 gene to be tested and identifying the genotype of the corresponding site, wherein the SNP site is the SNP site at position 124741207, position 124741301 and / or position 124741312 located on chromosome 15 of the porcine reference genome, and the porcine reference genome is Sscrofa11.1.

[0018] Preferably, the amplification primer design template may refer to the following region: 15:124739985-15:124741985. In one embodiment of the present invention, the nucleotide sequence of the primer is the sequence shown in SEQ ID No. 2 or 3 in the sequence listing.

[0019] In a fifth aspect, the present invention provides a detection kit utilizing the SNP molecular marker described in the first aspect. The kit comprises primers for specifically amplifying a SNP site in the promoter region of the porcine ACSL3 gene to be tested and identifying the genotype of the corresponding SNP site, wherein the SNP site is one or more of the SNP sites at positions 124741207, 124741301, and 124741312 on chromosome 15 of the porcine reference genome; the porcine reference genome is Sscrofa11.1, the SNP site at position 124741207 is a G>C mutation, the SNP site at position 124741301 is a C>T mutation, and the SNP site at position 124741312 is an A>C mutation (i.e., the SNP site is one or more of 15:124741207G>C, 15:124741301C>T, and 15:124741312A>C).

[0020] In a sixth aspect, the present invention provides a method for utilizing the aforementioned SNP markers to assist in the selection of superior PRRSV-resistant pigs. During breeding, the SNP molecular marker is preferably a CCA haplotype, which can improve the population's resistance to PRRSV. Specifically, the method includes selecting CCA haplotypes as both parents or a single parent for crossbreeding through molecular marker detection, and selecting offspring with the CCA haplotype for breeding.

[0021] The beneficial effects of the present invention are:

[0022] The present invention uses a genome resequencing method to search for and discover the ACSL3 gene promoter region variant site 15:124741312A>C related to PRRSV resistance and variant sites 15:124741207G>C and 15:124741301C>T with high linkage in a PRRSV artificially infected resource population; by identifying the genotypes of these sites, the mutant gene carrying status of the sites related to PRRSV resistance can be determined, and molecular markers of the corresponding sites can be used to quickly screen out individuals for constructing an excellent PRRSV-resistant population, providing a detection technology means for early selection and improving breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Heat map for SNP linkage analysis in the ACSL3 gene promoter region of the population.

[0024] Figure 2is the SNP haplotype frequency of the ACSL3 gene promoter region in the population.

[0025] Figure 3 This is a partial sequence of the sequencing results of the ACSL3 different haplotype promoter reporter gene vector.

[0026] Figure 4 The effect of SNPs on the transcriptional activity of ACSL3 promoter.

[0027] Figure 5 To analyze the protein expression and gray value after interfering with ACSL3 expression. DETAILED DESCRIPTION

[0028] In order to more clearly illustrate the purpose and technical solutions of the present invention, the following will briefly introduce the embodiments and drawings, and further explain the present invention in detail. The embodiments are only used to explain the present invention and are not used to limit the present invention.

[0029] Example 1

[0030] Analysis of the promoter region of the porcine ACSL3 gene by whole-genome resequencing

[0031] 1. Fresh whole blood was collected from 86 PRRSV-infected individuals. White blood cells were separated using red blood cell lysis buffer. White blood cell DNA was extracted using the phenol-chloroform method and sent to a sequencing company for whole genome sequencing.

[0032] 2. Use BWA software to perform whole genome sequence alignment of the pig reference genome 11.1.

[0033] 3. Use GATK, Beagle and other software to detect and annotate all SNP sites in all samples.

[0034] 4. All SNPs in the promoter region of the ACSL3 gene (default range is 2kb upstream) were extracted, and a total of 30 SNP sites were extracted. The extracted SNPs were imported into Haploview software to analyze the degree of linkage disequilibrium between SNPs. It was found that the 27th, 28th, and 29th SNP sites of the ACSL3 gene formed an LD module ( Figure 1 ), the haplotypes were CCA, GCA, GCC, and GTC, with frequencies of 0.450, 0.211, 0.203, and 0.118, respectively ( Figure 2 ).

[0035] The main haplotype in resistant individuals was CCA (81.8%), while the main haplotypes in susceptible individuals were CCA (42.9%) and GTC (42.9%) (Table 1).

[0036] Table 1 Haplotype distribution of ACSL3 promoter in resistant and susceptible individuals

[0037]

[0038] Example 2

[0039] Effects of SNPs in the Promoter Region of the Porcine ACSL3 Gene on Gene Expression

[0040] 1. In the whole genome sequencing data, CCA and GTC individuals were selected and used as PCR templates for amplification (Table 2).

[0041] Table 2 Primer sequence information

[0042]

[0043] 2. The region containing the 27th, 28th, and 29th SNP sites was selected as the target fragment. The target fragment was ligated to the pGL3-basic vector plasmid using homologous recombination, and homologous sequences were inserted into both sides of the target fragment using primers (Table 3).

[0044] Table 3 Basic information of ACSL3 dual luciferase plasmid construction

[0045]

[0046] 3. PCR amplification was performed in the individual using the above primers. The PCR reaction system was as shown in Table 4, and the total volume was 50 μL.

[0047] Table 4 PCR reaction system

[0048]

[0049]

[0050] The PCR reaction procedure is as follows:

[0051] 98℃2min

[0052] 98℃10s

[0053]

[0054] 4. PCR products were detected by 1.0% agarose gel electrophoresis at 150 V for 20 min and then recovered using an agarose gel recovery kit (FastPure Gel DNA Extraction Mini Kit, Vazyme).

[0055] 5. After double digestion of the pGL3-basic vector plasmid with KpnI and HindIII restriction enzymes, ligate with the target fragment, transform, culture the single clone overnight, and send to a sequencing company to check whether the sequence is correct ( Figure 3 ).

[0056] 6. Use Omega's Plasmid Midi Kit to remove endotoxins from the plasmid extraction kit according to the instructions to extract the dual-luciferase expression vector plasmid for transfection of different cells.

[0057] 7.293T and PK15 CD163 Cells were cultured in complete medium containing 10% fetal bovine serum and 1% double-streptomycin (penicillin + streptomycin) and grown in a 37°C, 5% CO2 incubator to 80-90% confluence. Cells were passaged into 24-well cell culture plates and transiently transfected when the monolayer confluence reached 70-80% under a microscope. Transfection was performed using Lipofectamine. TM 2000 reagent instructions. Dilute the empty plasmid, internal reference plasmid and recombinant vector into 50μL Opti-MEM medium respectively and mix gently; at the same time, dilute an appropriate amount of Lipo2000 into 50μL Opti-MEM medium, mix well, and incubate for 5 minutes; mix the above two evenly and incubate for 20 minutes; 293T and PK15 CD163 The cells were washed with PBS and 500 μL of Opti-MEM medium was added. 100 μL of the above transfection mixture was added dropwise to the washed cell wells and the culture plate was gently shaken to mix them evenly. After 6 h, the medium was changed to maintenance medium containing 2% fetal bovine serum. After further culture for 24 h, the cells were harvested and dual-luciferase activity was detected.

[0058] 8. Add 100 μL of 1× Passive Lysis Buffer (5× Passive Lysis Buffer diluted with PBS) to each well and shake on a horizontal shaker at room temperature for 30 minutes to fully lyse the cells. Take 10 μL of cell lysate to a 96-well plate, first add 50 μL of firefly luciferase substrate Luciferase Assay Substrate (diluted with Luciferase Assay Buffer II), use EnSpire multi-mode plate reader to measure the firefly luciferase activity value, record and save the data. Then add 50 μL of sea rennin luciferase substrate 1× Stop & Substrate (using Stop & Buffer dilution 50×Stop& The activity of Renilla luciferase was measured using an EnSpire multi-mode plate reader, and the data were recorded and saved.

[0059] 9. GraphPad Prism 10 was used for plotting and statistical analysis. Data are expressed as mean ± standard error. * < 0.05 indicates significant difference, and ** < 0.01 indicates extremely significant difference.

[0060] 10. The results showed that in 293T and PK15 CD163 The activity of CCA haplotype on ACSL3 gene promoter was significantly lower than that of GTC haplotype on ACSL3 gene promoter in cells ( Figure 4 ).

[0061] The above experimental analysis shows that, for the molecular marker of the present invention (SEQ ID NO. 1), the CCA haplotype can effectively reduce the expression of ACSL3 and plays an important role in regulating the expression of ACSL3.

[0062] Example 3

[0063] Effect of Interference on ACSL3 Gene Expression on Inhibition of PRRSV Proliferation

[0064] 1. Design three siRNAs for different interference targets of the ACSL3 gene, as shown in Table 5.

[0065] Table 5: ACSL3 gene siRNA sequences

[0066]

[0067] 2.PK15 CD163 Cells were cultured in complete medium containing 10% fetal bovine serum and 1% double-streptomycin (penicillin + streptomycin) and grown in a 37°C, 5% CO2 incubator to 80-90% confluence. Cells were passaged into 6-well cell culture plates and transiently transfected when the monolayer confluence reached 70-80% under a microscope. Transfection was performed using Lipofectamine. TM 2000 reagent instructions. Dilute NC and siRNA into 200μL Opti-MEM medium respectively and mix gently; at the same time, dilute an appropriate amount of Lipo2000 into 200μL Opti-MEM medium, mix well, and incubate for 5 minutes; mix the above two evenly and incubate for 20 minutes; PK15 CD163The cells were washed with PBS and 2 mL of Opti-MEM medium was added. 400 μL of the transfection mixture was added dropwise to the washed cell wells and the culture plate was gently shaken to mix evenly. After 6 h, the medium was changed to maintenance medium containing 2% fetal bovine serum. After 24 h of culture, the cells were infected with PRRSV for 48 h.

[0068] 3. Discard the cell culture medium, wash the cells with PBS, aspirate the PBS, add cell lysis buffer (200 μL per well), and place the cell culture plate on ice for 30 minutes to ensure complete lysis. Use a cell scraper to scrape the lysed cells, transfer them to a 1.5 mL centrifuge tube, and centrifuge at 12,000 rpm / min for 10 minutes. Discard the sticky nucleic acid material, and the remaining protein sample is added to the protein sample. Boil for 10 minutes to denature the protein, and store in a refrigerator at -20°C.

[0069] 4. Detect the protein concentration of all samples according to the instructions of the BCA protein concentration assay kit (P0012) of Biyuntian Company.

[0070] 5. After preparing the polyacrylamide gel, transfer it to an electrophoresis tank and add 1x electrophoresis buffer. Use a pipette to draw up the sample along the wall, insert it into the sample well, and slowly add the sample. Run electrophoresis at 80V for 30 minutes, then at 120V for 1 hour. Terminate the electrophoresis and transfer to the membrane. Cut the region containing the target gene according to the size of the protein maker and place it in transfer buffer. Cut the PVDF membrane into pieces that match the size of the gel, soak it in methanol for 3 minutes, and then soak it in transfer buffer for 5 minutes. Clamp the filter paper, gel, membrane, and filter paper together in the transfer tank and transfer at 250mA for 1 hour. Place the membrane in blocking buffer containing 5% skim milk powder at room temperature for 2 hours. After blocking, gently rinse the PVDF membrane twice with TBST and incubate in diluted primary antibody overnight at 4°C. After the primary antibody incubation, wash the membrane three times with TBST for 10 minutes each. Incubate the membrane in diluted secondary antibody at room temperature for 2 hours, and then wash the membrane three times with TBST for 10 minutes each. Mix equal volumes of developer A and developer B, add to the membrane and let it stand for 1 min, then place it in a chemiluminescence imaging analyzer for photographing and preservation.

[0071] 6. Grayscale values ​​of protein bands were calculated using Image J. GraphPad Prism 10 was used for plotting and statistical analysis. Data are expressed as mean ± standard error. * < 0.05 indicates significant difference, and ** < 0.01 indicates extremely significant difference.

[0072] 7. Western Blot results showed that compared with the NC group, the expression of ACSL3 protein in the ACSL3 siRNA transfection group was significantly downregulated, and the level of PRRSV N protein was also significantly downregulated ( Figure 5 ).

[0073] It can be seen from Examples 1 and 2 that in breeding, when the CCA haploid is selected for the molecular marker (SEQ ID NO.1 in the sequence list), the expression of ACSL3 protein can be significantly downregulated, thereby improving the resistance to PRRSV. When infected with PRRSV, the level of PRRSV N protein can be significantly downregulated, thereby achieving the effect of resisting porcine blue ear disease.

[0074] Based on the above, the molecular markers of the present invention can be used for assisted breeding of pigs, namely assisted breeding of blue ear disease-resistant pigs. Based on the molecular markers disclosed in the present invention, those skilled in the art can determine the genotype by sequencing a fragment containing one of bases 1223, 1317, or 1328 of SEQ ID NO.1, or amplify the relevant sequence using specific primers, perform enzyme digestion on the relevant sites, and then identify the genotype by gel electrophoresis.

[0075] During assisted breeding, parents with the CCA haplotype of the molecular marker of the present invention are selected for breeding, and offspring containing the CCA haplotype trait are selected.

Claims

1. A method for detecting molecular markers associated with porcine PRRSV resistance in the ACSL3 gene and its application in marker-assisted selection breeding of pigs, characterized in that: The molecular markers are SNP sites at positions 124741207, 124741301 and 124741312 located on chromosome 15 of the pig reference genome. The SNP site at position 124741207 is a G>C mutation, the SNP site at position 124741301 is a C>T mutation, and the SNP site at position 124741312 is an A>C mutation. These three sites were detected, and the CCA haplotypes of these three sites were dominant haplotypes. The selective breeding is the breeding of pig varieties resistant to blue ear disease, and the pig reference genome is Sscrofa11.

1.

2. Application of a method for detecting SNPs in a candidate genomic region associated with pig PRRSV resistance in pig marker-assisted selection breeding, characterized in that: The following steps are involved: Using the pig genomic DNA to be tested as a template, the ACSL3 gene promoter region fragment was amplified by PCR, and the genotypes of the following SNP sites located in the ACSL3 gene promoter region were identified: SNP sites at positions 124741207, 124741301 and 124741312 located on chromosome 15 of the pig reference genome, the SNP site at position 124741207 is a G>C mutation, the SNP site at position 124741301 is a C>T mutation, and the SNP site at position 124741312 is an A>C mutation. The CCA haplotypes of the three sites are dominant haplotypes. The selective breeding is the breeding of pig varieties resistant to blue ear disease, and the pig reference genome is Sscrofa11.

1.

3. Use of primers for specifically amplifying SNP sites related to pig PRRSV resistance in the promoter region of the pig ACSL3 gene to be tested and identifying the genotype of the corresponding site, or a kit containing the primers in pig marker-assisted selective breeding, wherein the SNP sites are SNP sites at positions 124741207, 124741301 and 124741312 located on chromosome 15 of the pig reference genome, the SNP site at position 124741207 is a G>C mutation, the SNP site at position 124741301 is a C>T mutation, and the SNP site at position 124741312 is an A>C mutation, the CCA haplotype of the three sites is a dominant haplotype, the selective breeding is the breeding of pig varieties resistant to blue ear disease, and the pig reference genome is Sscrofa11.

1.

4. The use according to claim 3, wherein The sequences of the primers are the sequences shown in SEQ ID No. 2 and 3 in the sequence listing.

5. A method for breeding pig varieties resistant to blue ear disease, which comprises selecting the SNP site at position 124741207 on chromosome 15 of the pig reference genome as a G>C mutation, the SNP site at position 124741301 as a C>T mutation, and the SNP site at position 124741312 as an A>C mutation, and selecting CCA haplotype pigs at these three sites for breeding, wherein the pig reference genome is Sscrofa11.1.

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