Porcine ACSL3 gene promoter region SNP marker and application thereof in porcine anti-PRRSV breeding

Abstract

The invention provides a pig ACSL3 gene promoter region SNP marker and application of the pig ACSL3 gene promoter region SNP marker in porcine reproductive and respiratory syndrome resistant breeding. According to the present invention, the SNPs in the ACSL3 gene mover region is analyzed by using the whole genome re-sequencing data to discover that the three mononucleotide variations on the pig ACSL3 gene mover are related to the porcine reproductive and respiratory syndrome disease resistance, and the CCA haplotype can improve the porcine reproductive and respiratory syndrome disease resistance of the population, such that the early individual selection can be performed by using the molecular marker; the method is used for auxiliary breeding of porcine reproductive and respiratory syndrome resistant varieties. The invention provides a reliable molecular marker for genetic improvement of porcine anti-PRRSV, and has important significance for breeding for disease resistance of pigs.

Description

Technical Field

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

[0002] 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 in pigs of all ages and reproductive disorders in sows. The PRRSV genome is highly variable and easy to recombine, which makes vaccine immunity unable to effectively inhibit its spread. Disease-resistant breeding enhances the host's resistance to PRRSV at the genetic level, fundamentally inhibiting the spread of PRRSV.

[0003] Fatty acids play an important role in physiological processes and participate in the formation of many lipids, including the formation of triacylglycerols and cholesterol esters, the formation of structural lipids such as phospholipids, conversion to alcohols or aldehydes, carbon addition and extension or carbon reduction and shortening, insertion or removal of double bonds, or covalent binding to proteins (Kuwata H and Hara S2019, Quan J et al 2021, Soupene E and Kuypers FA 2008). Before free fatty acids enter the above metabolic processes, they must be converted into activated forms of fatty acids by forming thioester bonds with coenzyme A under the catalysis of members of the acyl-coenzyme A synthetases (ACSs) family. With the completion of genome sequencing of multiple species, 26 confirmed ACS family members have been detected using highly conserved amino acid sequence motifs (Watkins PA et al 2007). Among them, long-chain acyl-CoA synthetase ACSL in the acyl-CoA synthetase family is a key enzyme for the synthesis of long-chain fatty acids. The ACSL family usually contains five members: ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6. The distribution sites of each member in the body are different, but they all have the function of activating long-chain fatty acids. ACSL3, also known as ACS3, is mainly located in the periphery of lipogenic cells and in the cytoplasm of the endoplasmic reticulum (Kassan A et al 2013, Poppelreuther M et al 2012). ACSL3 has a high substrate specificity for lauric acid, myristic acid, and eicosapentaenoic acid ethyl ester, and can preferentially activate saturated fatty acids and unsaturated fatty acids (Fujino et al 1996).

[0004] As the study of ACSL3 deepens, it is found that its biological role is extensive. Li et al. found that ACSL3 can play an important role as a negative regulator in the process of ferroptosis after brain injury through the GPX4-ACSL3 axis (Li M et al 2022). After using targeted siRNA to interfere with the expression of ACSL3 in Huh7 cells, the secretion of apolipoprotein B, the main component of lipoprotein, was inhibited, lipoproteins were rapidly degraded, and the secretion of HCV virus particles in host cells was also inhibited (Nchoutmboube JA et al2013). Yao et al. found that the replication efficiency of poliovirus was significantly downregulated after treating cells with siRNA targeting ACSL3 (Yao H and Ye J 2008). In addition, ACSL3 also has relatively complex functions in different development stages of different types of cancer, such as participating in the synthesis and deposition of lipids in the early stages of carcinogenesis, and increasing lipid utilization in the late stages of breast cancer and prostate cancer (Tang Y et al 2018, Wright et al 2017).

[0005] So far, there are no reports on SNP markers in the promoter region of the porcine ACSL3 gene mediating resistance to blue ear disease in pigs. Therefore, studying the SNPs of the ACSL3 gene and identifying its relationship with blue ear disease resistance can provide a favorable theoretical basis for molecular marker-assisted breeding and facilitate the rapid 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 a PRRSV artificially infected resource population, and explores the genetic variation in the promoter region of the porcine ACSL3 gene to explore molecular markers and detection methods related to porcine blue ear disease resistance, and applies them to pig genetic breeding, establishes a porcine molecular marker-assisted breeding method, and provides gene markers and technical methods for the selection and breeding of porcine individuals resistant to blue ear disease.

[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 the nucleotide sequence thereof is SEQ ID NO.1. The molecular marker is obtained by resequencing the whole genome of a PRRSV artificially infected resource population, obtaining sequencing data, extracting all SNPs in the ACSL3 gene promoter region, and determining that three highly linked SNP sites in the promoter region of the porcine ACSL3 gene, namely 15:124741207, 15:124741301 and 15:124741312 (the reference genome of the pig is Sscrofa11.1) may be related to blue ear disease resistance. Those skilled in the art should understand 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 of the sequence shown in SEQ ID NO.1, which fragment 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 fragments of the three sites of the 1223rd base, the 1317th base and the 1328th base.

[0009] Further, the present invention combines frequency distribution and linkage disequilibrium analysis to extract three variant sites located in the ACSL3 promoter region, namely 15:124741207, 15:124741301 and 15:124741312 as candidate sites. The variant types of the three candidate sites are identified as follows: the SNP site at position 124741207 on chromosome 15 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.

[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 the proliferation of PRRSV.

[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 an application of the SNP molecular marker described in the first aspect in pig genetic breeding for blue ear disease resistance. The SNP site is a SNP site 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, the SNP site is one or more of the SNP sites located 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 primers for specifically amplifying SNP sites 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 sites are SNP sites at positions 124741207, 124741301 and / or 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 can 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 and 3 in the sequence listing.

[0019] In a fifth aspect, the present invention provides a detection kit using the SNP molecular marker as described in the first aspect. The kit comprises primers for specifically amplifying the SNP site in the promoter region of the porcine ACSL3 gene to be detected 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 using the above-mentioned SNP marker to assist in breeding superior pigs resistant to PRRSV, wherein the SNP molecular marker is preferably a CCA haplotype during breeding, which can improve the disease resistance of the group to PRRSV. Specifically, it includes selecting CCA haplotype as both parents or a single parent for hybrid breeding through the molecular marker detection, and selecting offspring with CCA haplotype for breeding.

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

[0022] The present invention searches for and discovers a variant site 15:124741312A>C in the promoter region of the ACSL3 gene related to PRRSV resistance and variant sites 15:124741207G>C and 15:124741301C>T with a high degree of linkage in a PRRSV artificially infected resource population through a genome resequencing method; the genotypes of these sites can be identified to determine the carrying status of the mutant genes at the sites related to PRRSV resistance, and individuals for constructing an excellent PRRSV resistance population can be quickly screened out using molecular markers at the corresponding sites, thereby providing a detection technology means for early breeding and improving breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[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 solution 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 porcine ACSL3 gene by whole genome resequencing

[0031] 1. Collect fresh whole blood from 86 PRRSV artificially infected test individuals, separate white blood cells using red blood cell lysis buffer, extract white blood cell DNA using the phenol chloroform method, and send it to a sequencing company for whole genome sequencing.

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

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

[0034] 4. All SNPs in the promoter region of the ACSL3 gene (the default range is 2kb upstream) were extracted, and a total of 30 SNP sites were extracted. The extracted SNPs were imported into the 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%), and the main haplotypes in susceptible individuals were CCA (42.9%) and GTC (42.9%) (Table 1).

[0036] Table 1 Distribution of ACSL3 promoter haplotypes in resistant and susceptible individuals

[0037]

[0038] Example 2

[0039] Effect of SNPs in the Promoter Region of 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, and the target fragment was connected to the pGL3-basic vector plasmid using the homologous recombination method, and the 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. The above primers were used to perform PCR amplification in individuals. 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. The PCR product was detected by 1.0% agarose gel electrophoresis at 150 V for 20 min, and then recovered by agarose gel recovery kit (FastPure Gel DNA Extraction Mini Kit, Vazyme).

[0055] 5. After double digestion with KpnI and HindⅢ restriction enzymes, the pGL3-basic vector plasmid was connected with the target fragment, transformed, cultured overnight with single clones, and sent to a sequencing company to check whether the sequence is correct ( Figure 3 ).

[0056] 6. Use Omega's Plasmid Midi Kit to remove endotoxins and 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 The cells were cultured in complete medium containing 10% fetal bovine serum and 1% double antibody (penicillin + streptomycin) and grown in a 37°C, 5% CO2 incubator to 80-90% confluence. The cells were subcultured to a 24-well cell culture plate and transiently transfected when the monolayer confluence reached 70-80% under a microscope. Transfection was performed according to 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 5min; mix the above two evenly, and incubate for 20min; 293T and PK15 CD163 The cells were washed with PBS, and 500 μL Opti-MEM medium was added. 100 μL of the above transfection mixture was added to the washed cell wells, and the culture plate was gently shaken to mix them evenly. After 6 hours, the medium was replaced with a maintenance medium containing 2% fetal bovine serum. After continuing to culture for 24 hours, 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 BufferII), 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 (Use Stop & Buffer dilution 50×Stop& Substrate, prepared on the spot), and the Renilla luciferase activity was measured using the EnSpire multi-mode plate reader, and the data were recorded and saved.

[0059] 9. GraphPad Prism 10 was used for drawing and statistical analysis. The data were expressed as mean ± standard error. * < 0.05 indicated significant difference, and ** < 0.01 indicated 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] Through the above experimental analysis, it can be known 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 interfering with ACSL3 gene expression on inhibiting PRRSV proliferation

[0064] 1. Design 3 siRNAs for different interference targets of ACSL3 gene, as shown in Table 5.

[0065] Table 5: ACSL3 gene siRNA sequences

[0066]

[0067] 2.PK15 CD163 The cells were cultured in complete medium containing 10% fetal bovine serum and 1% double antibody (penicillin + streptomycin) and grown in a 37°C, 5% CO2 incubator to 80-90% confluence. The cells were subcultured to a 6-well cell culture plate and transiently transfected when the monolayer confluence reached 70-80% under a microscope. Transfection was performed according to 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 5min; mix the above two evenly, and incubate for 20min; PK15 CD163The cells were washed with PBS, and 2 mL of Opti-MEM medium was added. 400 μL of the above transfection mixture was added to the washed cell wells, and the culture plate was gently shaken to mix them evenly. After 6 hours, the medium was replaced with a maintenance medium containing 2% fetal bovine serum. After 24 hours of culture, the cells were infected with PRRSV for 48 hours.

[0068] 3. Discard the cell culture medium, wash the cells with PBS, aspirate the PBS, add cell lysis solution (200 μL per well), and place the cell culture plate on ice for 30 minutes to ensure full lysis. Use a cell scraper to scrape the lysed cells, transfer them to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm / min for 10 minutes, discard the sticky nucleic acid material, and the remaining is the protein sample. Add 5× protein loading buffer to the protein sample, boil for 10 minutes to denature the protein, and store in a -20°C refrigerator.

[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 the polyacrylamide gel is prepared, move it into the electrophoresis tank and add 1× electrophoresis buffer. Use a pipette to absorb the sample against the wall, insert it into the sample well and slowly add the sample. Perform electrophoresis at 80V for 30min, and 120V for 1h. Then terminate the electrophoresis and transfer the membrane. Cut the region where the target gene is located according to the size of the protein Maker and place it in the transfer solution. Cut the PVDF membrane into the same size as the gel, soak it in methanol for 3min, and soak it in the transfer solution for 5min. Clamp the filter paper, gel, membrane, and filter paper together and place them in the transfer tank. Transfer the membrane at 250mA for 1h. Place the membrane in a blocking solution containing 5% skim milk powder and block it at room temperature for 2h. After blocking, gently rinse the PVFD membrane twice with TBST, place it in the diluted primary antibody, and incubate it overnight at 4℃. After the primary antibody incubation is completed, wash the membrane 3 times with TBST, each time for 10min. Incubate the membrane in the diluted secondary antibody at room temperature for 2h, and wash the membrane 3 times with TBST, each time for 10min. Mix equal volumes of developer A and developer B, add to the membrane and let stand for 1 min, then put into 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, and data were expressed as mean ± standard error, * < 0.05 indicated significant differences, and ** < 0.01 indicated extremely significant differences.

[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 significantly downregulated accordingly ( 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 table), the expression of ACSL3 protein can be significantly down-regulated, thereby improving the resistance to PRRSV. When infected with PRRSV, the level of PRRSV N protein can be significantly down-regulated, 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, that is, 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 the 1223rd base, the 1317th base, or the 1328th base of the SEQ ID NO.1 sequence, or amplify the relevant sequence by specific primers, perform enzyme digestion on the relevant sites, and then identify the genotype by gel electrophoresis.

[0075] In 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 molecular marker method for the ACSL3 gene associated with porcine PRRSV resistance, characterized in that: The molecular marker is 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 are detected, and the CCA haplotype of these three sites is the dominant haplotype.

2. A molecular marker associated with resistance to blue ear disease in pigs, which is a fragment of the nucleotide sequence shown in SEQ ID NO.1, and the fragment includes at least one of the 1223rd base, the 1317th base or the 1328th base of the sequence shown in SEQ ID NO.

1.

3. A method for detecting SNPs in a candidate genomic region associated with pig PRRSV resistance, comprising the following steps: 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 / or 124741312 located on chromosome 15 of the pig reference genome, the SNP site at position 124741207 was a G>C mutation, the SNP site at position 124741301 was a C>T mutation, and the SNP site at position 124741312 was an A>C mutation.

4. The method according to claim 3, characterized in that The CCA haplotypes of these three loci were selected as the dominant haplotypes for disease resistance.

5. Primers used to specifically amplify the SNP sites in the promoter region of the porcine ACSL3 gene to be tested and related to porcine PRRSV resistance and to identify the genotypes of the corresponding sites, wherein the SNP sites are the SNP sites at positions 124741207, 124741301 and / or 124741312 located on chromosome 15 of the porcine reference genome, and the porcine reference genome is Sscrofa11.

1.

6. The primer according to claim 5, characterized in that The sequences thereof are those shown in SEQ ID No. 2 and 3 in the sequence listing.

7. A detection kit comprising the primers according to claim 5 or 6.

8. Use of the molecular marker method according to claim 1, the molecular marker according to claim 2, the detection method according to claim 3 or 4, the primer according to claim 5 or 6, or the kit according to claim 7 in marker-assisted selection breeding of pigs.

9. The use according to claim 8, characterized in that The pig marker-assisted selection breeding is the breeding of SNP-marked pig varieties resistant to blue ear disease.

10. A method for breeding a pig variety 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 breeding the CCA haplotype pigs at these three sites.

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