A DNA methylation molecular marker for detecting pig PRRSV resistance and its application

Abstract

The present invention belongs to the technical field of breeding of resistant varieties, and more specifically, relates to a DNA methylation molecular marker for detecting pig PRRSV resistance and its application. The molecular marker is a sequence rich in CpG dinucleotides in the promoter region of pig miR-212, which is shown in SEQ ID NO.1. All C bases in the sequence are converted into T bases, and then TG is replaced with CG to obtain the molecular marker, whose nucleotide sequence is shown in SEQ ID NO.2. The present invention designs DNA methylation primers for the miR-212 promoter region sequence through the aforementioned molecular markers, which can be used to effectively, accurately and quantitatively detect the degree of DNA methylation of the pig miR-212 promoter region sequence. The DNA methylation molecular marker for pig PRRSV resistance provided by the present invention can be used as a new epigenetic molecular marker, providing a theoretical basis and genetic basis for molecular breeding of pigs with PRRS resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of resistant variety breeding, and more specifically, relates to a DNA methylation molecular marker for detecting pig PRRSV resistance and an application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is caused by the porcine blue ear virus (PRRSV) and is one of the most serious infectious diseases in the swine industry, causing significant economic losses. Despite extensive work in vaccine development, biosafety, and swine health, PRRSV's highly susceptible genome mutation and recombination, immunosuppression, antibody-dependent enhancement, and persistent infection make its prevention, control, and eradication extremely difficult. In recent years, with the deepening of PRRSV research both domestically and internationally, it has been increasingly recognized that identifying target genes associated with PRRSV susceptibility / resistance and breeding PRRSV-resistant pigs can be important means of controlling PRRSV, with significant practical significance for the prevention and control of swine diseases.

[0003] Whether pigs develop disease is a function of the interaction between pathogen invasion and the body's defenses. Genetic factors play a significant role in the development of disease, and different populations and individuals exhibit varying resistance and susceptibility to the same pathogen. Both PRRS and African swine fever outbreaks share common characteristics: despite high morbidity and mortality rates, some resistant breeds and individuals exist. Studies have shown that indigenous Chinese pig breeds are highly resistant to PRRSV, while foreign breeds are susceptible. Meishan pigs, however, exhibit greater resistance to PRRSV than Landrace pigs.

[0004] DNA methylation is one of the earliest discovered, stably inherited epigenetic modifications. Studies have shown that DNA methylation plays an important role in viral infection. For example, PRRSV can limit the high expression of the transcription factor IRF3 through DNA methylation modification, thereby inhibiting the antiviral effect mediated by type I interferon. In a study on the resistance of Chinese native pigs to Escherichia coli F18, it was found that the expression of the FUT3 gene was significantly higher in some susceptible individuals of Sutai pigs and Meishan pigs than in those with strong resistance. The molecular mechanism is that two methylation sites in the promoter region of the FUT3 gene affect the binding of transcription factors (SP1 and HIF1A) to the FUT3 promoter.

[0005] Therefore, it has become a technical problem that needs to be solved urgently to conduct research on the differential characteristics of pig growth groups with significant differences in transcriptome, genome and epigenetics based on the genetic information of the pig genome, and to obtain a molecular marker of DNA methylation that can be used in pig disease-resistant breeding and then obtain excellent genetic traits and excellent varieties (lines). Summary of the Invention

[0006] In order to overcome the defects of the prior art, the purpose of the present invention is to provide a DNA methylation molecular marker for detecting pig PRRSV resistance and its application.

[0007] In the first aspect, the present invention provides a DNA methylation molecular marker for detecting pig PRRSV resistance, wherein the molecular marker is a CpG dinucleotide-rich sequence in the pig miR-212 promoter region, as shown in SEQ ID NO.1. All C bases in the sequence are converted into T bases, and then TG is replaced with CG to obtain the molecular marker, whose nucleotide sequence is shown in SEQ ID NO.2.

[0008] In a second aspect, the present invention provides a primer pair for the molecular marker, wherein the upstream and downstream primers of the primer pair are shown in SEQ ID NO. 3-4, respectively.

[0009] In a third aspect, the present invention provides a method for detecting DNA methylation of pig PRRSV resistance, comprising the following steps:

[0010] S1, extracting the pig genomic DNA to be tested;

[0011] S2, bisulfite conversion followed by DNA purification;

[0012] S3, using the purified DNA as a template, performing PCR amplification using the primer pair;

[0013] S4. Sequence the amplified product of S3 to quantitatively detect the methylation level of the miR-212 promoter region sequence.

[0014] In a fourth aspect, the present invention provides the use of the molecular marker in assisting pig breeding against PRRSV.

[0015] In a fifth aspect, the present invention provides a breeding method for improving pig resistance to PRRSV, wherein the method is used to detect the methylation degree of the pig miR-212 promoter region sequence, and individuals with low methylation levels are screened out as breeding parents.

[0016] The beneficial effects of the present invention are:

[0017] Using the aforementioned molecular markers, the present invention designed DNA methylation primers targeting the miR-212 promoter region sequence, which can be used to effectively, accurately, and quantitatively detect the degree of DNA methylation in the porcine miR-212 promoter region sequence. The DNA methylation molecular markers for porcine PRRSV resistance provided by the present invention can serve as novel epigenetic molecular markers, providing a theoretical basis and genetic foundation for molecular breeding of PRRS-resistant pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The methylation levels of miR-212 in Meishan pigs are shown in Figure 2. Solid circles represent methylation, and hollow circles represent unmethylation.

[0019] Figure 2 The methylation levels of miR-212 in Landrace pigs are shown in Figure 2. Solid circles represent methylation, and hollow circles represent unmethylation.

[0020] Figure 3 is the expression level of miR-212 in the lung tissues of Meishan and Landrace pigs, and the ordinate is the relative expression level of miR-212.

[0021] Figure 4 The vertical axis represents the expression level of miR-212 before and after PRRSV infection, and the vertical axis represents the relative expression level of miR-212.

[0022] Figure 5 Figure 2 shows the effect of miR-212 on PRRSV replication. A shows the inhibition of PRRSV replication by miR-212 mimic, with the ordinate representing the PRRSV copy number; B shows the inhibition of PRRSV replication by miR-212 inhibitor, with the ordinate representing the PRRSV copy number; C shows the inhibition of PRRSV N protein expression by miR-212 mimic; D shows the inhibition of PRRSV N protein expression by miR-212 inhibitor. DETAILED DESCRIPTION

[0023] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0024] Example 1: Extraction and quality identification of porcine genomic DNA

[0025] DNA was extracted using the phenol / chloroform method. The specific steps are as follows:

[0026] (1) Take 0.1g (0.5cm3) of lung tissue from one-month-old Meishan and Changbai mice and chop them into small pieces. Place in a glass homogenizer, add 1ml of cell lysis buffer and homogenize until no tissue pieces are seen. Transfer to a 1.5ml centrifuge tube, add 20μl of proteinase K (500ug / ml), and mix well. Place in a 65℃ constant temperature water bath for 30min, or transfer to a 37℃ water bath for 12-24h, intermittently shaking the centrifuge tube several times. Centrifuge at 12000rpm in a desktop centrifuge for 5min, and take the supernatant into another centrifuge tube.

[0027] (2) Add 2 volumes of isopropanol and mix thoroughly by inversion. Threads can be seen. Use a 100 μl pipette to remove the fragments, let them air dry, and then re-dissolve them in 200 μl of TE. (PCR reactions can be performed. If further purification is required, follow the steps below.)

[0028] (3) Add equal amounts of phenol, chloroform, and isoamyl alcohol (25:24:1), shake well, and centrifuge at 12,000 rpm for 5 min.

[0029] (4) Transfer the upper layer of solution to another tube, add equal volumes of chloroform and isoamyl alcohol (24:1), shake to mix, and centrifuge at 12000 rpm for 5 min.

[0030] (5) Transfer the upper layer of solution to another tube, add 1 / 2 volume of 7.5 mol / L ammonium acetate and 2 times the volume of anhydrous ethanol, mix well, and precipitate at room temperature for 2 minutes. Centrifuge at 12000 rpm for 10 minutes.

[0031] (6) Carefully pour off the supernatant and place the centrifuge tube upside down on absorbent paper to remove any remaining droplets attached to the tube wall.

[0032] (7) Wash the precipitate once with 1 ml of 70% ethanol and centrifuge at 12000 rpm for 5 min.

[0033] (8) Carefully pour off the supernatant, place the centrifuge tube upside down on absorbent paper, remove any remaining droplets attached to the tube wall, and dry at room temperature.

[0034] (9) Add 200 μl of TE to re-dissolve the precipitate, and then use a Thermo NANO Drop 2000 Spectrophotometer to measure the concentration and purity of the extracted DNA. After the test, the qualified DNA samples were stored at -20°C.

[0035] Example 2: Amplification of the CpG island in the porcine miR-212 promoter region

[0036] 1. Bisulfite treatment of DNA samples

[0037] To convert DNA, use the EpiTec Bisulfite Handbook (59104). First, add 30 mL of absolute ethanol to Buffer BW and mix thoroughly by inversion before use. Then, add 27 mL of absolute ethanol to Buffer BD and mix thoroughly by inversion before use. Equilibrate the DNA sample and all buffers to room temperature.

[0038] Bisulfite conversion of DNA:

[0039] (1) Add 800 μL of RNase-free water to each tube of Bisulfite Mix and shake until completely dissolved. You can also dissolve it in a 60°C water bath.

[0040] (2) Preparation of the reaction system, see Table 1. Note: The total volume of DNA solution and RNase-free water must reach 20 μL.

[0041] Table 1 DNA conversion reaction system

[0042]

[0043]

[0044] (3) Cover the lid, mix the reaction system, and wait for it to turn from green to blue at room temperature before proceeding to the next step.

[0045] (4) Perform bisulfite DNA conversion on a PCR instrument, using the procedure shown in Table 2.

[0046] Table 2 DNA conversion reaction procedure

[0047]

[0048] Note: “-” means it can be left overnight

[0049] 2. Purification of converted DNA

[0050] (1) Transfer the converted DNA solution to a 1.5 mL centrifuge tube, add 560 μL of BL Buffer, and mix thoroughly.

[0051] (2) Transfer the DNA solution to EpiTec spin columns, centrifuge at 14,000 rpm for 1 min at room temperature, discard the filtrate in the collection tube, and return the EpiTec spin columns to the collection tube.

[0052] (3) Add 500 μL of Buffer BW to each EpiTec spin column, centrifuge at 14,000 rpm for 1 min at room temperature, discard the filtrate in the collection tube, and return the EpiTec spin columns to the collection tube.

[0053] (4) Add 500 μL of Buffer BD to each EpiTec spin column, place at room temperature for 15 min, centrifuge at 14,000 rpm for 1 min at room temperature, collect the filtrate in the tube, and return the EpiTec spin columns to the collection tube.

[0054] (5) Add 500 μL of Buffer BW to each EpiTec spin column, centrifuge at 14,000 rpm for 1 min at room temperature, collect the filtrate in the tube, and return the EpiTec spin columns to the collection tube.

[0055] (6) Repeat step (5) once.

[0056] (7) Place the EpiTec spin columns in a new 2 mL centrifuge tube and centrifuge at 14,000 rpm for 1 min at room temperature to remove any residual liquid.

[0057] (8) Place the EpiTec spin columns in a new 1.5 mL centrifuge tube and incubate in a 56°C water bath for 5 min.

[0058] (9) Place the EpiTec spin columns in a new 1.5 mL tube, add an appropriate amount of EB to the center membrane of the EpiTec spin columns, centrifuge at 14,000 rpm for 1 min at room temperature, and freeze the resulting DNA solution at -20°C.

[0059] 3. PCR amplification

[0060] First, all C in the analyzed sequence (shown in SEQ ID NO.1) was converted to T, and then TG was potentially replaced with CG. The sequence after replacement is shown in SEQ ID NO.2. Primers were designed using Primer 5.0. The primer information is shown in Table 3.

[0061] Table 3 Methylation amplification primer information

[0062]

[0063] The CpG island of the miR-212 gene promoter was amplified by PCR using sulfite-treated genomic DNA as a template. The specific reaction system and procedure were as follows: SYBR qRT-PCR Master Mix 12.5 μL, Forward primer / Reverse primer 0.3 μL, DNA 1 μL, ddH2O up to 25.0 μL; the reaction procedure was 95°C for 2 min, 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 72°C for 10 min, 35 cycles.

[0064] 4. Detection and recovery of PCR amplification products

[0065] Take 5 μL of PCR amplification product and mix it with a small amount of Loading Buffer. Detect the PCR product amplification results by 1.5% agarose gel electrophoresis, and recover the target band using the gel recovery kit produced by Biotek.

[0066] A. Add the PCR amplified product completely to the wells of the agarose gel. After the bands are completely separated by electrophoresis, cut the gel containing the target fragment under long-wave ultraviolet light and place it into a clean 1.5 mL centrifuge tube.

[0067] B. Add 200-400 μL of sol-binding solution according to the size of the gel block;

[0068] C. Sol in a 56°C water bath until the gel is dissolved. Shake the centrifuge tube every 1-2 minutes during the process to accelerate dissolution.

[0069] D. Aspirate the liquid obtained in step C into the adsorption column and centrifuge at 12000 rpm for 1 minute;

[0070] E. Discard the waste liquid, add 700 μL of rinse solution to the adsorption column, and centrifuge at 12000 rpm for 1 minute;

[0071] F. Discard the waste liquid, place the adsorption column into a new centrifuge tube, and centrifuge at 12000 rpm for 2 minutes;

[0072] G. Place the adsorption column in another new 1.5 mL centrifuge tube, add 25 μL of ddH2O to the center of the adsorption membrane, incubate at 37°C for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute. The liquid obtained in the centrifuge tube is the purified product and should be stored at -20°C.

[0073] 5. Connection and transformation of recovered products with cloning vectors

[0074] (1) Ligation reaction

[0075] The cloning vector used in this experiment was pMD18-T. The ligation system was as follows, and the reaction was carried out at 16°C for 3 h.

[0076] pMD18-T Vector 1μL

[0077] Solution I 5μL

[0078] 4 μL of product was recovered;

[0079] (2) Transformation of ligation products

[0080] A. Pipette 5 μL of the ligation product onto a clean bench and add it to a 1.5 mL centrifuge tube containing 50 μL of competent cells. Mix thoroughly and place on ice for 30 minutes.

[0081] B. Heat shock at 42°C for 1.5 minutes, followed by ice bath for 2 minutes;

[0082] C. Add 400 μL of LB liquid medium without Amp and incubate at 37°C, 220 rpm, and shake for 60 min.

[0083] D. Centrifuge at 6000 rpm for 6 minutes to collect cells, leaving 80 μL of upper culture medium;

[0084] E. Pipette the cells evenly and spread them on the prepared LB solid medium. Incubate at 37°C for 1 hour, then invert the plate and culture overnight.

[0085] (3) Screening and sequencing of positive clones

[0086] A. On a clean bench, use a sterilized pipette tip to pick single colonies from a plate cultured overnight and place them in a 1.5 mL centrifuge tube to which 400 μL of LB liquid medium containing Amp has been added. Pick 6 single colonies per plate.

[0087] B. Incubate at 37°C, 220 rpm, and shake for 4 h.

[0088] C. Use 1 μL bacterial solution as template and amplify with original specific primers or universal primers of pMD18-T. Detect by electrophoresis and screen positive clones for testing.

[0089] D. Compare and analyze the sequencing results to identify the methylation level of the miR-212 promoter region sequence in different pig breeds.

[0090] Example 3: Methylation analysis of CpG islands in the promoter region of miR-212 in Meishan pigs and Landrace pigs

[0091] The target fragment amplified in Example 2 was recovered and sequenced after T-linking. A total of 35 CG sites were detected in the 382 bp PCR product. At least 10 single clones were tested for each sample, and the correct sequencing results were submitted to the online software QUMA (http: / / quma.cdb.riken.jp / ) for methylation statistical analysis and corresponding graphs. The methylation sequencing results showed that the CpG island in the promoter region of miR-212 in Meishan pig lung tissue had relatively few methylated CG sites (0.44%, Figure 1 ), while the demethylated CG sites of miR-212 promoter in Landrace pigs were more than those in Meishan pigs (8.9%, Figure 2A study injected PRRSV into 28-day-old Meishan and Landrace pigs intramuscularly. The results showed that Meishan pigs had stronger resistance than Landrace pigs, while Landrace pigs were significantly more susceptible to PRRSV than Meishan pigs (Xiao Qiang, 2010). Therefore, it is speculated that the methylation level of the miR-212 promoter region in Landrace pigs is higher than that in Meishan pigs, resulting in lower expression of miR-212 in Landrace pigs compared to Meishan pigs, which in turn affects the differences in PRRSV resistance among different pig breeds.

[0092] Example 4: Detection of miR-212 expression levels in Meishan pigs and Landrace pigs, and before and after PRRSV infection

[0093] RNA was extracted from lung tissue samples of one-month-old Meishan and Landrace pigs, as well as total RNA from Marc-145 cells before and after PRRSV infection, using the TRIZOL method. miR-212 cDNA was reverse transcribed using the stem-loop method, and the expression levels of miR-212 in Meishan and Landrace pigs, as well as before and after infection, were detected by RT-qPCR. U6 was used as an internal reference. The sequences of the quantitative primers and stem-loop primers are shown in Table 4. The results showed that the expression level of miR-212 in lung tissue of Meishan pigs was significantly higher than that in Landrace pigs ( Figure 3 ), after PRRSV infection, the expression level of miR-212 was significantly decreased.

[0094] Table 4. Primer sequences for miR-212 quantitative detection

[0095]

[0096] Example 5: miR-212 inhibits PRRSV replication

[0097] Seeding: cultured Marc-145 cells were plated at 1×10 5 6-well plates were inoculated with the number of cells, and 2 mL of DMEM (high glucose culture medium) containing 10% fetal bovine serum was added to each well and cultured at 37° C. and 5% CO 2 .

[0098] Lipofectamine transfection: When the cell density reaches 70%-80%, replace the cells with serum-free OPTI medium (purchased from Invitrogen) and place them in an incubator for transfection. Prior to transfection, dilute the miR-212 mimic, miR-212 inhibitor, and the control NC mimic and NC inhibitor in OPTI. Prepare a mixture of 10 μL of miRNA and 250 μL of OPTI per well. Simultaneously, prepare a mixture of 8 μL of transfection reagent Lip2000 and 250 μL of OPTI per well. Incubate at room temperature for 5 minutes. Then, mix the two dilutions and incubate at room temperature for 20 minutes before transfection. Add 500 μL of the prepared mixture to each well and bring the total volume to 2 mL with OPTI. 4-6 hours after transfection, replace the DMEM medium with the cells and infect them with PRRSV at an MOI (multiplicity of infection) of 0.1. Allow to adsorb for 1 hour before replacing the cells with normal culture medium.

[0099] Table 3 Sequences of synthetic miRNAs mimics and inhibitors

[0100]

[0101] PRRSV copy number detection: Cells were collected 36 hours after infection with PRRSV, and total cell RNA and total protein were extracted. The extracted total cell RNA was reverse transcribed into cDNA. PRRSV replication was detected by absolute fluorescence quantitative PCR. The absolute fluorescence quantitative system was 10 μL: 5 μL SYBR Green I realtime PCR Mix, 0.15 μL each of upstream primer ORF7-F and downstream primer ORF7-R, 1 μL cDNA, 0.4 μL probe-ORF7 and 3.3 μL double dehydrated. The probe (GenBank: HM853673.2) and primer sequences are shown in Table 4 below:

[0102] Table 4 Primers used for qRT-PCR

[0103]

[0104] The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 20 s, annealing at 64°C for 20 s, extension at 72°C for 15 s and collection of fluorescence signals, for 40 cycles.

[0105] The results showed that after 36h of PRRSV infection, the relative expression of miR-212 was significantly decreased ( Figure 4 ), transfection of miR-212mimic could significantly (p<0.05, Figure 5 A) inhibited the copy number of PRRSV, whereas miR-212 inhibitor could significantly (p<0.05, Figure 5B) increased the copy number of PRRSV. Western Blot results showed that miR-212 could significantly inhibit the expression of PRRSV N protein ( Figure 5 C, 5D). This is consistent with the verification results at the mRNA level, indicating that miR-212 can significantly inhibit the proliferation of PRRSV.

[0106] Example 6: Application of DNA methylation molecular markers for detecting pig PRRSV resistance in assisting pig PRRSV resistance breeding, comprising the following steps:

[0107] S1, extracting the pig genomic DNA to be tested;

[0108] S2, bisulfite conversion followed by DNA purification;

[0109] S3. Using the purified DNA as a template, PCR amplification was performed using the primer pair shown in SEQ ID NO. 3-4;

[0110] S4. Sequencing the amplified product of S3 to quantitatively detect the methylation degree and differentially methylated sites of the miR-212 promoter sequence;

[0111] Individuals with low methylation levels were selected as breeding parents.

[0112] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0113] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A DNA methylation molecular marker for detecting pig PRRSV resistance, characterized in that: The molecular marker is a CpG dinucleotide-rich sequence in the promoter region of pig miR-212, which is shown in SEQ ID NO.

1. All C bases in the sequence are converted into T bases, and then TG is replaced with CG to obtain the molecular marker, whose nucleotide sequence is shown in SEQ ID NO.

2.

2. A primer pair for amplifying the molecular marker according to claim 1, characterized in that: The upstream and downstream primers of the primer pair are shown in SEQ ID NO.3-4 respectively.

3. The use of the molecular marker according to claim 1 in assisting pig anti-PRRSV breeding, characterized in that: The application comprises the following steps: S1, extracting the pig genomic DNA to be tested; S2, bisulfite conversion followed by DNA purification; S3. Using the purified DNA as a template, perform PCR amplification using the primer pair described in claim 2; S4. Sequence the amplified products to detect the methylation sites and methylation levels of the miR-212 promoter region sequence, and select individuals with low methylation levels as breeding parents.

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