Bcl2l15 gene molecular marker for resisting porcine reproductive and respiratory syndrome and application thereof

Abstract

The application provides a molecular marker related to porcine reproductive and respiratory syndrome resistance in a BCL2L15 gene and application thereof, the molecular marker rs331890123 is located at a 3'UTR region of a porcine BCL2L15 gene on a 106681000th base of a chromosome 4 of a porcine reference genome. The application takes the rs331890123 as a detection site, finds that the site polymorphism significantly influences the expression of the BCL2L15 gene, and further inhibits the proliferation of a porcine reproductive and respiratory syndrome virus. The application also establishes a kind of molecular marker polymorphism Tetra-primer ARMS-PCR rapid detection method related to porcine reproductive and respiratory syndrome resistance. The application provides a new SNP molecular marker for marker-assisted selection of porcine reproductive and respiratory syndrome disease resistance breeding.

Description

Technical Field

[0001] This invention belongs to the field of pig molecular breeding technology, specifically relating to a molecular marker related to resistance to porcine reproductive and respiratory syndrome (PRRS) and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), causing huge economic losses to the global pig industry. The main clinical manifestations of PRRS are reproductive disorders in sows, such as premature birth, stillbirth, and mummified fetuses, as well as respiratory disorders in pigs of all ages, including dyspnea, coughing, and fever. PRRSV is a single-stranded RNA virus with high variability and rapid recombination characteristics, leading to poor vaccine efficacy and potential threats such as vaccine strain infection and virulence reversion. Furthermore, PRRSV infection can cause host immunosuppression, resulting in persistent viral infection and secondary infections with other pathogens. Therefore, enhancing the host's resistance at the genetic level is an effective way to control PRRS and a key focus of pig genetic breeding improvement efforts.

[0003] Studies have shown that local Chinese pigs, such as Tongcheng pigs and Dapulian pigs, exhibit strong resistance to porcine reproductive and respiratory syndrome (PRRS) (Jiang et al., 2013; Liang et al., 2016). The inventors' research group, through artificial infection experiments with highly pathogenic PRRSV, found that Tongcheng pigs showed stronger resistance to PRRS compared to Large White pigs. Clinical symptoms, histopathological damage, and serum viral load in Tongcheng pigs infected with PRRSV were significantly lower than in Large White pigs (Liang et al., 2016). While blood viral load and other traits after PRRSV infection can directly reflect an individual's antiviral capacity, this indicator can only be detected after PRRSV infection. If molecular markers significantly correlated with blood viral load in pigs infected with PRRSV can be detected, resistant individuals can be screened for disease-resistant breeding. Summary of the Invention

[0004] To address the aforementioned technical issues, this invention analyzes whole-genome sequencing data from Tongcheng pigs and Large White pigs, discovering that SNP polymorphisms in the 3'UTR region of the porcine BCL2L15 gene are correlated with the expression level of PRRSV resistance genes, thereby affecting the inhibition of PRRSV proliferation. Consequently, SNP sites in the 3'UTR region of the porcine BCL2L15 gene can serve as molecular markers for screening PRRS-resistant individuals, providing a novel molecular breeding marker for porcine reproductive and respiratory syndrome (PRRS) resistance breeding.

[0005] One of the purposes of the invention is to provide a molecular marker associated with resistance to porcine reproductive and respiratory syndrome (PRRS) located at the SNP site in the 3'UTR region of the BCL2L15 gene, namely the rs331890123 site, where the allele mutation of the nucleotide sequence at position 258, R, is A or G, as shown in SEQ ID NO.1.

[0006] Furthermore, the present invention provides a nucleic acid sequence for the molecular marker, which is a fragment whose nucleotide sequence is the nucleotide sequence shown in SEQ ID NO.1 containing the base at position 264. Generally, this fragment can be amplified by PCR and then used for sequencing, detection by electrophoresis, or detection by a probe. For example, in the present invention, a 431bp amplification product is obtained by amplification using the BCL2L15 outer primer pair (nucleotide sequences such as SEQ ID NO.6 and SEQ ID NO.7). Of course, this fragment is not limited to this; any fragment that meets the detection conditions and contains the rs331890123 site is acceptable.

[0007] Furthermore, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1.

[0008] A second objective of this invention is to provide specific primers for the specific amplification of the aforementioned molecular markers. In one embodiment of this invention, the primers comprise a BCL2L15 outer primer pair and a BCL2L15 inner primer pair for amplifying the sequence shown in SEQ ID NO.1. The nucleotide sequences of the BCL2L15 outer primer pair are shown in SEQ ID NO.6 and SEQ ID NO.7, and the nucleotide sequences of the BCL2L15 inner primer pair are shown in SEQ ID NO.8 and SEQ ID NO.9. These primers can be used alone or together. For example, when used alone, amplification using the BCL2L15 outer primer pair can be performed by sequencing to obtain a fragment containing the rs331890123 site. The two methods are used in combination. Based on the amplification results of the upstream primer of the BCL2L15 outer primer and the downstream primer of the BCL2L15 inner primer, and combined with the amplification results of the downstream primer of the BCL2L15 outer primer and the upstream primer of the BCL2L15 inner primer, the size of the amplified fragment is detected by electrophoresis to determine the rs331890123 site.

[0009] A third objective of this invention is to provide a detection reagent or detection kit containing the aforementioned specific primers.

[0010] Preferably, it further includes one or more of the following reagents: Taq enzyme, dNTPs, MgCl2, PCR buffer, double-distilled water; or it further includes one or more of the following reagents: Taq DNA MasterMix, double-distilled water.

[0011] A fourth objective of this invention is to provide the application of the aforementioned molecular markers, specific primers, reagents, or kits in pig assisted breeding. The base R at position 264 of the nucleotide sequence shown in SEQ ID NO.1 being A indicates a low viral load after PRRSV infection. In assisted breeding, pigs with a homozygous genotype of AA (base R at position 264 of the nucleotide sequence shown in SEQ ID NO.1) and / or a heterozygous genotype of AG are selected for breeding. Preferably, pigs with a homozygous AA genotype are selected for breeding.

[0012] The fifth objective of this invention is to provide a method for breeding pigs resistant to porcine reproductive and respiratory syndrome (PRRS), which involves selecting pigs with a homozygous genotype of AA at the rs331890123 locus and / or a heterozygous genotype of AG for breeding. Preferably, pigs with a homozygous genotype of AA at the rs331890123 locus are selected for breeding. That is, individuals with the A allele at the molecular marker rs331890123 locus and a homozygous AA genotype have stronger resistance to PRRSV infection. By selecting individuals with the AA genotype at rs331890123 for breeding, which exhibit strong resistance to PRRSV, the disease resistance of the herd can be improved, which is beneficial to disease-resistant pig breeding.

[0013] Preferably, the method uses sample pig DNA as a template and performs PCR amplification using the inner and outer primer pairs shown in SEQ ID NO. 6-9. If there are three amplified fragments with sizes of 431bp, 289bp, and 197bp, the individual genotype of the sample to be tested is determined to be heterozygous AG; if there are two amplified fragments with sizes of 431bp and 289bp, the individual genotype of the sample to be tested is determined to be homozygous AA; and if the sizes are 431bp and 197bp, the individual genotype of the sample to be tested is determined to be homozygous GG.

[0014] The sixth objective of this invention is to provide a method for detecting molecular markers (SNPs) associated with porcine reproductive and respiratory syndrome (PRRS) resistance using the aforementioned detection kit, comprising the following steps:

[0015] Step 1: Extract genomic DNA from the sample to be tested;

[0016] Step 2: Using the DNA extracted in Step 1 as a template, construct the Tetra-primer ARMS-PCR amplification system using the inner and outer primers shown in SEQ ID NO. 6-9 and perform PCR amplification.

[0017] Step 3: Perform electrophoresis detection and result determination on the PCR amplification products.

[0018] The method described above, preferably, determines the result as follows:

[0019] In BCL2L15 gene detection, if there are three amplified fragments with sizes of 431bp, 289bp, and 197bp, the individual genotype of the sample is determined to be heterozygous AG; if there are two amplified fragments with sizes of 431bp and 289bp, the individual genotype of the sample is determined to be homozygous AA; and if the sizes are 431bp and 197bp, the individual genotype of the sample is determined to be homozygous GG.

[0020] The beneficial effects of this invention are as follows:

[0021] The molecular marker rs331890123, associated with porcine reproductive and respiratory syndrome (PRRS) resistance, provided by this invention, is located in the 3'UTR region of the porcine BCL2L15 gene and has not been reported in porcine disease resistance genetic analysis. The AA genotype at this locus can increase BCL2L15 gene expression and effectively inhibit PRRSV proliferation. This invention provides a novel molecular genetic marker for PRRS resistance breeding and marker-assisted selection, and provides its application in identifying PRRS resistance and assisting breeding, as well as disclosing a detection kit for detecting molecular markers associated with PRRS resistance. Attached Figure Description

[0022] Figure 1 The image shows the dual-luciferase results of gene expression regulation by different genotype vectors of the molecular marker rs331890123, which is associated with resistance to porcine reproductive and respiratory syndrome (PRRS) provided by this invention.

[0023] Figure 2 The Western blot results and grayscale analysis of different genotype vectors of the molecular marker rs331890123, which is associated with resistance to porcine reproductive and respiratory syndrome (PRRS), provided by this invention, inhibit PRRSV proliferation. The Control vector is pcaggs-flag-n.

[0024] Figure 3 The image shows the agarose gel electrophoresis pattern of the Tetra-primer-ARMS-PCR amplification PCR product of the molecular marker rs331890123, which is associated with porcine reproductive and respiratory syndrome (PRRS) resistance, provided by this invention. Lane M is the DNA molecular weight marker (DL1000), and lane H is the blank control. Detailed Implementation

[0025] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0026] Example 1: Application of the molecular marker rs331890123 associated with porcine reproductive and respiratory syndrome (PRRS) resistance in regulating BCL2L15 gene expression.

[0027] 1.1 RNA extraction and reverse transcription

[0028] Leukocyte samples frozen at -80℃ were used to extract RNA using the Trizol lysis method. RNA concentration was measured using a Nanodrop 2000 nucleic acid and protein analyzer, and the RNA was stored at -80℃ for later use. Using the TaKaRa PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time), the volume of 1 μg of RNA was first calculated based on the concentration, and then the reaction solution was prepared according to the following system; the reaction was carried out at 42℃ for 2 min to remove genomic DNA.

[0029]

[0030]

[0031] Then, the first strand of cDNA was synthesized, and the reaction system was prepared as follows:

[0032]

[0033] The reaction procedure was: 42℃ for 15 min, 85℃ for 5 s.

[0034] 1.2 Construction of different genotype plasmids of the molecular marker rs331890123 associated with porcine reproductive and respiratory syndrome (PRRS) resistance

[0035] Primers were designed based on the sequence information of the BCL2L15 gene in the NCBI database (Table 1). Individuals with different genotypes of rs331890123 were selected to amplify the gene transcript sequence.

[0036] Table 1. Basic information on the construction of the BCL2L15 dual-luciferase plasmid.

[0037]

[0038] 1.3 PCR amplification

[0039] The PCR reaction mixture (20 μL) consisted of: 10.0 μL of 2×Taq Master Mix, 0.6 μL each of forward and reverse primers (10 μM), 2.0 μL of DNA template (30 ng / μL), and 6.8 μL of ddH2O. The PCR program was: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 55℃ annealing for 17 s, and 72℃ extension for 30 s, for a total of 35 cycles; followed by storage at 72℃ for 5 min and then at 4℃. The amplified products were detected by 1.5% agarose gel electrophoresis, and the electrophoresis results were observed using a gel imaging system. The agarose gel was then recovered and stored using a FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0040] 1.4 Plasmid Construction and Extraction

[0041] The psi-check2 plasmid and the corresponding target fragment were double-digested using restriction enzyme sites (XhoⅠ and NotⅠ) to obtain sticky ends. The plasmid vector DNA and the inserted DNA fragment were mixed to prepare a 5 μl DNA solution, followed by the addition of an equal volume of T4 DNA ligase. The mixture was incubated at 16°C for 30 min. The reaction solution was then added to 100 μl of competent cells for transformation and plated. Single clones were picked and sent to the company for sequencing for subsequent experiments. Dual-luciferase plasmids for different genotypes were extracted using the Plasmid Midi Kit endotoxin-free plasmid extraction kit.

[0042] 1.5 Dual-luciferase reporter gene assay

[0043] PK-15 CD163 Cells were seeded into 24-well plates and transfected when the cell confluence reached approximately 80%. Transfection was performed according to the Lipofectamine™ 2000 reagent instructions. Six hours after transfection, the transfection solution was removed, and 2% complete culture medium was added for further culture. After 24 hours, the culture medium was removed, and the cells were washed 1-2 times with PBS. 100 μL of 1×Passive Lysis Buffer (5×Passive Lysis Buffer diluted with ddH2O, freshly prepared) was added, and the cells were shaken horizontally at room temperature for 30 minutes to induce complete cell lysis. 10 μL of cell lysis buffer was transferred to 96-well microplates. 50 μL of Luciferase Assay Substrate (diluted with Luciferase Assay Buffer II, freshly prepared) was added, and the Luciferase activity was measured using an EnSpire multi-mode plate reader. The data were recorded and saved. Then, 50 μL of 1×Stop& Luciferase Substrate was added. Substrate (using Stop&) Buffer diluted 50×Stop& Substrate (prepared fresh for use) was used to measure the renin-luciferase activity using an EnSpire multi-mode plate reader. Data were recorded and saved. Firefly luciferase activity was used as an internal control, and the ratio of renin-luciferase activity to firefly luciferase activity was used to reflect the regulation of gene expression. GraphPad Prism 10 was used for plotting and statistical analysis. *<0.05 indicates significant difference, **<0.01 indicates highly significant difference.

[0044] The results showed that different genotypes of the molecular marker rs331890123, associated with resistance to porcine reproductive and respiratory syndrome (PRRS), were associated with the expression of the BCL2L15 gene. Compared with the GG genotype, the expression level of the BCL2L15 gene was significantly increased in the AA genotype. Figure 1 ).

[0045] Example 2: Application of the molecular marker rs331890123 associated with porcine reproductive and respiratory syndrome (PRRS) resistance in inhibiting viral replication.

[0046] 2.1 Plasmid Construction

[0047] Primers were designed based on the sequence information of the BCL2L15 gene in the NCBI database (Table 2). Based on individual samples of different genotypes from Example 1, gene transcript sequence plasmids and corresponding target fragments were amplified. Then, sticky ends were obtained by restriction enzyme digestion, followed by ligation and transformation using ligase. Single clones were then selected and sent to the company for sequencing for subsequent experiments. The Plasmid Midi Kit was used to extract gene expression vector plasmids for different genotypes for transfection into different cell types.

[0048] Table 2. Basic information on the construction of the BCL2L15 expression plasmid.

[0049]

[0050] 2.2 Viral protein detection

[0051] Marc145 cells were seeded into 6-well plates and transfected when the cell confluence reached approximately 80% according to the Lipofectamine™ 2000 reagent instructions. Six hours after transfection, the transfection medium was removed, and 2% complete culture medium was added for further culture. After 24 hours, the culture medium was removed, and the cells were washed 1-2 times with PBS. Cells were then infected with PRRSV for 48 hours before protein extraction. Protein concentration in all samples was determined according to the Beyotime BCA protein assay kit (P0012). Protein expression was then detected by SDS-PAGE electrophoresis.

[0052] The results showed that the molecular marker rs331890123, associated with porcine reproductive and respiratory syndrome (PRRS) resistance, can affect the expression of the BCL2L15 gene, thereby affecting the inhibition of viral replication. In BCL2L15 gene expression, the AA genotype was significantly higher than the GG genotype, with the AA genotype expressing 1.6 times more viral load. In viral protein detection, the viral load in cells overexpressing the BCL2L15 gene was significantly downregulated, with the AA genotype downregulated by 0.4 times and the GG genotype by 0.3 times; the AA genotype was lower than the GG genotype. Figure 2The results indicate that rs331890123 can affect viral load after PRRSV infection. Previous studies have shown that the expression level of the BCL2L15 gene can effectively affect porcine reproductive and respiratory syndrome virus (PRRSV) replication (Bai et al., 2019), which is consistent with our findings. In disease-resistant breeding applications, individuals with the AA genotype can be preferentially selected for breeding to improve the disease resistance of the population.

[0053] Example 3: Establishment of a Tetra-primer-ARMS-PCR method for detecting molecular marker polymorphisms associated with porcine reproductive and respiratory syndrome (PRRS) resistance.

[0054] 3.1 Extraction of porcine genomic DNA

[0055] Take a tissue sample the size of a soybean, cut it into small pieces with ophthalmic scissors, and extract porcine genomic DNA from the tissue using the commonly used phenol-chloroform crude extraction method or other recognized methods with the same efficacy, and use it as a template for PCR amplification.

[0056] 3.2 Primer Design

[0057] Primers for molecular markers were designed using the Tetra-primer ARMS-PCR online design program (http: / / cedar.genetics.soton.ac.uk / public_html / primer1.html). Two inner primers with 3′ ends paired with the two alleles of the SNP and extending in opposite directions, and two outer primers extending in opposite directions, were designed for the SNP sites (Table 3). Simultaneously, a mismatch was artificially introduced at the 3rd base of the 3′ end of the inner primer to increase amplification specificity (Table 4, underlined letters represent mismatched bases). Primer specificity was tested using the NCBI BLAST program.

[0058] Table 3 Basic Information on SNP Sites

[0059]

[0060] SNP ID: Ensembl genome browser database; Reference genome: Sscrofa11.1

[0061] Table 4 Primer sequence information

[0062]

[0063] 3.3 Tetra-primer ARMS-PCR amplification

[0064] The PCR reaction system (20 μL) consisted of: 10.0 μL of 2×Taq Master Mix, 0.8 μL each of two outer primers (10 μM), 1.2 μL each of two inner primers (10 μM), 2.0 μL of DNA template (30 ng / μL), and 6.0 μL of ddH2O. The PCR program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56.9℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 30 cycles; followed by storage at 72℃ for 5 min and then at 4℃. The amplified products were detected by 1.5% agarose gel electrophoresis. The electrophoresis results were observed and saved using a gel imaging system. Different genotypes were distinguished based on the size of the amplified fragments and the number of bands.

[0065] 3.4 Results

[0066] The Tetra-primer ARMS-PCR amplification system includes one pair of outer primers and one pair of inner primers. The target fragment amplified by the outer primer pair (SEQ ID NO. 6-7) (as shown in SEQ ID NO. 1) serves as a positive control, amplifying the largest fragment (431 bp). In SEQ ID NO. 1, the letter R indicates an A or G allele mutation in rs331890123. The inner primers specifically bind to the mutant site G (SEQ ID NO. 8) and the wild-type site A (SEQ ID NO. 9), respectively. SEQ ID NO. 9 and outer primer SEQ ID NO. 6 amplify a target band of 289 bp, while SEQ ID NO. 8 and outer primer SEQ ID NO. 7 amplify a target band of 197 bp. The size and presence of the electrophoretic bands can determine whether a mutation has occurred and the individual's genotype.

[0067] Method for determining the SNP site polymorphism detection results of rs331890123:

[0068] If there are three amplified fragments with sizes of 431, 289, and 197 bp respectively, then the individual's genotype is determined to be heterozygous AG.

[0069] If there are two amplified fragments, with sizes of 431 bp and 289 bp respectively, then the individual's genotype is determined to be homozygous AA.

[0070] If there are two amplified fragments, 431 bp and 197 bp in size respectively, then the individual's genotype is determined to be homozygous GG; the result is as follows. Figure 3 As shown.

[0071] This invention establishes a rapid Tetra-primer ARMS-PCR detection method for rs331890123, which requires only one step of PCR and electrophoresis to identify the genotype of an individual. The SNP site of rs331890123 can serve as a molecular marker associated with porcine reproductive and respiratory syndrome (PRRS) resistance, providing a theoretical basis and technical support for subsequent analysis of the molecular mechanism of PRRSV resistance in pigs and for disease-resistant breeding in pigs.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of specific primers for specifically amplifying molecular markers related to porcine reproductive and respiratory syndrome (PRRS) resistance, or detection reagents or kits containing said specific primers, in the preparation of products for identifying PRRS resistance or PRRS-resistant breeding products, wherein the molecular marker related to PRRS resistance is an allelic mutation of the base A or G at position 264 of the nucleotide sequence shown in SEQ ID NO.1; when used in PRRS-resistant breeding, pigs with a homozygous genotype where the base R at position 264 of the nucleotide sequence shown in SEQ ID NO.1 is AA are selected for breeding.

2. Use according to claim 1, wherein The primers are a BCL2L15 outer primer pair and a BCL2L15 inner primer pair. The nucleotide sequences of the BCL2L15 outer primer pair are shown in SEQ ID NO.6 and SEQ ID NO.7, and the nucleotide sequences of the BCL2L15 inner primer pair are shown in SEQ ID NO.8 and SEQ ID NO.

9.

3. The use according to claim 1, wherein The detection reagent or detection kit also includes one or more of the following reagents: Taq enzyme, dNTPs, MgCl2, PCR buffer, and double-distilled water; Alternatively, it may include one or more of the following reagents: Taq DNA MasterMix, double-distilled water.

4. A method for breeding pigs resistant to porcine reproductive and respiratory syndrome (PRRS), wherein pigs with a homozygous genotype whose nucleotide sequence shown in SEQ ID NO.1 has a base R of AA at position 264 are selected for breeding.

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