SNP mutation site of porcine MyoG gene and its application

Abstract

The present invention discloses a SNP mutation site of the porcine MyoG gene, which is a SNP rs3471653254C>T mutation located at the upstream promoter of the porcine MyoG gene and 299bp before the transcription start site. The present invention also provides the application of the SNP mutation site in breeding for improving the lean meat yield performance of pigs. The SNP mutation site SNP rs3471653254C>T of the present invention is significantly correlated with the differentiation efficiency of primary myoblasts of pigs into muscle, and can be used as a single base editing site for gene editing. Without introducing any exogenous DNA into the genome, the local pig breeds can maintain the "purity" of the genome, which can not only retain excellent traits to the maximum extent, but also specifically improve its lean meat rate, greatly improving the production and yield of high-quality pork.

Description

Technical Field

[0001] The invention belongs to the field of biomolecular breeding, and in particular relates to a SNP mutation site of a pig MyoG gene and an application thereof. Background Art

[0002] Domestic pigs (Sus scrofa domesticus) are one of the most important livestock for human protein sources. Local Chinese pigs, such as the Guangdong-Guangxi Small-eared Pig, Ningxiang Pig, and Taoyuan Black Pig, are popular among consumers for their excellent meat quality and unique flavor. Unfortunately, due to the lack of strict and systematic specialized breeding, most of the important economic traits of local Chinese pigs, especially growth rate and meat production, are far lower than those of lean pigs, which is an important obstacle to their further industrial development. However, although the traditional breeding method based on quantitative genetics has been successful in the breeding of lean pigs, it has also taken more than a century, which is obviously no longer appropriate in today's rapidly developing biotechnology.

[0003] In recent years, with the rise of molecular biology, especially the introduction and continuous updating of gene editing technology, the progress of traditional breeding has been greatly accelerated, and animal breeding experts have also focused more on accelerating the production of lean meat in pigs through molecular breeding. Before applying molecular breeding, it is necessary to screen and identify specific sites that can significantly affect lean meat yield or growth rate. The types of these sites include single nucleotide polymorphisms (SNPs), gene copy number variations (CNVs), insertions and deletions (InDels), etc., located on genes or their promoters, or intergenic regions or non-coding regions. They are different in the two major pig breeds of Chinese local pigs and Western lean pigs and are significantly related to production performance. They are increasingly used in livestock breeding and have made some important progress. For example, mutations in the Myostatin gene can lead to a significant increase in the meat production of Meishan pigs. It can be seen that identifying gene sites that can significantly affect lean meat production will be a key breakthrough point for rapidly increasing the meat production of local pigs.

[0004] Single nucleotide polymorphism (SNP) is an important basis for studying the genetic variation of animal strains. With its known, heritable, detectable and wide distribution, it is widely used in population genetics research and disease-related gene research. The distribution of SNP is regionally specific and population-specific, which may be one of the reasons for the phenotypic differences between populations. In addition, SNP is widely distributed in the pig genome, and through whole genome resequencing, it was found that there are many different SNPs in the important myogenic marker protein MyoG gene between Western lean pigs and Chinese local pigs. So far, many studies have shown that SNP is related to the formation of skeletal muscle, but little is known about its role and molecular mechanism in causing differences in meat production between breeds. If a certain SNP site in pigs can be identified as a key factor affecting important economic traits of pigs, the information of the SNP site can be used to simulate natural mutations through gene editing or marker-assisted selection, or even whole genome selection, to accelerate the genetic progress of traits, thereby cultivating new high-yield and high-quality pig breeds, improving the social pig farming efficiency, and reducing consumption. Summary of the invention

[0005] One object of the present invention is to provide a SNP mutation site that can effectively improve the differentiation efficiency of porcine myoblasts in response to the above key problems to be solved.

[0006] Another object of the present invention is to provide an application of the SNP mutation site.

[0007] In order to achieve the above-mentioned invention object, the present invention provides a SNP mutation site of the porcine MyoG gene, wherein the SNP mutation site is a SNP rs3471653254C>T mutation located at the upstream promoter of the porcine MyoG gene and 299 bp before the transcription start site. The site is a C base in Chinese local pigs and a T base in Western lean pigs. The mutation site does not involve amino acid mutations and is a synonymous mutation.

[0008] Preferably, the SNP mutation site is located at position 296 of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] Preferably, the SNP mutation site affects the myogenic differentiation efficiency of porcine primary myoblasts.

[0010] On the other hand, the present invention also provides the application of the SNP mutation site of the pig MyoG gene in breeding for improving pig lean meat yield performance, wherein the SNP mutation site is the SNP rs3471653254C>T mutation located at the upstream promoter of the pig MyoG gene and 299bp before the transcription start site.

[0011] On the other hand, the present invention also provides the use of a reagent for detecting the SNP mutation site of the pig MyoG gene in breeding for improving pig lean meat yield performance, wherein the SNP mutation site is the SNP rs3471653254C>T mutation located at the upstream promoter of the pig MyoG gene and 299 bp before the transcription start site.

[0012] Specifically, the present invention relates to utilizing the SNP rs3471653254C>T mutation of the porcine MyoG gene to improve the myogenic differentiation efficiency of porcine primary cells and thus improve the lean meat production performance of pigs. When the SNP rs3471653254 site in the MyoG gene recombinant expression plasmid is a T base, the myogenic differentiation level of porcine primary myoblasts is higher. The myoblast differentiation efficiency detection indicators include the fusion rate and differentiation rate of myotube differentiation and the protein and mRNA expression of myogenic marker genes.

[0013] This invention uses pig primary myoblasts as the research object, clarifies that the SNP rs3471653254C>T of the MyoG gene is significantly correlated with the differentiation efficiency of pig primary myoblasts, reveals the economic production value of SNP rs3471653254C>T, and provides a reference for the feasibility of further using SNP rs3471653254C>T as a single base editing site to improve pig production performance. In addition, using SNPs from different pig breeds for single base editing mutations can enable local pig breeds to maintain the "purity" of the genome without introducing any exogenous DNA into the genome. On the basis of maximally retaining the excellent traits of Chinese local pig breeds such as high-quality meat quality, it can overcome the disadvantage of low meat production, specifically improve its lean meat rate, and significantly improve the production and output of high-quality pork, providing new ideas for molecular breeding that takes into account both lean meat amount and meat quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the sequencing peak graph result of SNP rs3471653254 site in two major pig breeds.

[0015] Figure 2 This is a schematic diagram of the two molecular mechanisms by which SNP rs3471653254C>T regulates myogenic differentiation.

[0016] Figure 3 This is a schematic diagram of the 11 SNPs screened and located in the MyoG gene of two major pig breeds.

[0017] Figure 4 It is the differentiation result of porcine primary myoblasts transfected with MyoG plasmids from two major pig breeds.

[0018] Figure 5The results of MyoG gene expression after overexpression of two MyoG plasmids in primary porcine myoblasts.

[0019] Figure 6 The expression results of MyoD and MyHC after overexpression of two MyoG plasmids in primary porcine myoblasts.

[0020] Figure 7 The results of ChIP-qPCR analysis of the enrichment of HOXA5 in the promoter region of the MyoG gene in two major pig breeds.

[0021] Figure 8 It is the result of the dual fluorescence reporter system analyzing the fluorescence activity of the promoter fragment where the SNP rs3471653254 site is a T base or a C base.

[0022] Fig. 9 It is a dual fluorescence reporter system to analyze the interaction between LncRNAmyoparr and miR-30b-3p through predicted binding sites and the expression trend of the two.

[0023] Fig.10 These are the differentiation results of porcine primary myoblasts treated with agonists and inhibitors of miR-30b-3p. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the reagents, instruments, and technical means of operation used in the examples are all reagents, instruments, or technical means of operation known in the art.

[0025] The present invention provides a SNP mutation site of the porcine MyoG gene, which is located upstream of the MyoG gene promoter and 299 bp before the transcription start site, also known as SNP rs3471653254. The site is a C base in Chinese local pigs and a T base in Western lean pigs. The mutation site does not involve amino acid mutations and is a synonymous mutation. Figure 1 The results of sequencing peaks of SNP rs3471653254 in two major pig breeds are shown, where GS represents Guangdong-Guangxi Small-eared Spotted Pig and DR represents Duroc Pig. The SNP mutation site is located at position 296 of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0026] The molecular mechanism of the above SNP rs3471653254C>T in regulating myogenic efficiency is as follows: After the SNPrs3471653254 site in the promoter region changes from C to T, it promotes the enrichment of transcription factor HOXA5 in the promoter, resulting in an increase in the transcription levels of MyoG and LncRNA myoparr. When the expression of LncRNA myoparr increases, it promotes LncRNAmyoparr to act as a sponge to absorb miR-30b-3p. After the expression of miR-30b-3p is reduced, the differentiation efficiency of myoblasts will be significantly improved. Figure 2 Schematic diagram of the two molecular mechanism pathways by which SNP rs3471653254C>T regulates myogenic differentiation.

[0027] Example 1: SNP rs3471653254C>T mutation affects myogenic differentiation of porcine primary myoblasts

[0028] 1.1 Culture and transfection of primary porcine myoblasts

[0029] According to the resequencing results, 11 SNPs were screened in two major pig breeds, Western lean pigs and Chinese local pigs (chr9:113785911-rs3471653254, chr9:113785870-rs3472810668, chr9:113784650-rs81415971, chr9:113783771-rs331549227, chr9:113783487-rs337830285, chr9: 113783432——rs326150042, chr9:113783272——rs338390679, chr9:113783266——rs329303654, chr9:113783008——rs333670668, chr9:113782960——rs327642157, chr9:113782885——rs322813478)) were synthesized to construct MyoG gene recombinant expression vectors ( Figure 3 , Figure 3 11 SNP sites have been annotated in the genome), including Western lean pigs (taking Duroc pigs as an example, hereinafter referred to as DR) and Chinese local pigs (taking Guangdong and Guangxi small-eared spotted pigs as an example, hereinafter referred to as GS).

[0030] The steps for constructing the MyoG gene recombinant expression vector are as follows: the full-length MyoG gene sequence (>NC_010451.4:c113786206-113782870, Sus scrofa isolate TJ Tabasco breed Duroc chromosome 9, Sscrofa11.1, whole genome shotgun sequence) was inserted into the vector, and the specific SNP sites were set as follows: Figure 3 As shown, the MyoG overexpression plasmid was obtained by inserting the protein into the EcoRI / NotI site of pcDNA3.1.

[0031] Primary porcine myoblasts were isolated from the dorsal muscle of embryonic day 35 and grown in DMEM medium containing 20% ​​fetal bovine serum. When the primary porcine myoblasts grew to a confluence of about 80%, the MyoG gene recombinant expression vector was transfected using liposome transfection. The cells were transfected with the same medium as above 24 hours after transfection and cultured at 5% CO2 and 37°C. Subsequently, 2 days after transfection and when the cells grew to a confluence of about 100%, the DMEM medium containing 2% horse serum was replaced to induce differentiation. After 5 days, the cells were collected for subsequent experiments.

[0032] 1.2 RNA extraction and qPCR

[0033] Collect primary porcine myoblasts to extract RNA. The reagents needed are TRIzol, chloroform, isopropanol, and 75% v / v ethanol prepared with water treated with diethyl pyrocarbonate (DEPC). The steps are as follows: (1) Add TRIzol lysis buffer to the culture dish to lyse the cells; (2) Add 0.2 mL of chloroform for every 1 mL of TRIzol, shake vigorously for 30 seconds, and let it stand at room temperature for 3 minutes; (3) Centrifuge at 12000×g for 15 minutes at 4℃ to separate the samples. The top aqueous phase is the layer where RNA is located; (4) Aspirate the sample in the aqueous phase, add isopropanol with the same volume as the aspirated liquid, and let it stand at -20℃ for 30 minutes; (5) Centrifuge at 12000×g at 4℃ for 15 minutes, and keep the gelatinous precipitate at the bottom; (6) Wash the RNA precipitate twice with 75% ethanol; (7) Add DEPC water to the EP tube to dissolve the RNA precipitate.

[0034] After RNA was extracted, a reverse transcription kit (StarScript III one-tube de-genomic reverse transcription kit A230-10, Beijing Kangrun Bio) was used to reverse transcribe RNA into cDNA for subsequent qPCR experiments.

[0035] 1.3 Protein extraction and Western Blot

[0036] Porcine primary myoblasts were collected to extract proteins, and the cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer with a final concentration of 1 nm phenylmethylsulfonyl fluoride (PMSF), and the protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. The expression of related proteins was detected using a Western Blot imaging system.

[0037] 1.4 Results and Analysis

[0038] After overexpressing two MyoG vectors, MyHC immunofluorescence staining experiments showed that the differentiation and fusion levels of primary myoblasts transfected with the MyoG plasmid of Western lean pigs were significantly higher, indicating that these SNP sites play an important role in regulating myogenesis. Figure 4 As shown, the differentiation results of porcine primary myoblasts after transfection with MyoG plasmids of two major porcine species are shown.

[0039] The results of quantitative PCR showed that the expression level of MyoG was significantly increased after overexpression of the two MyoG plasmids, and this trend was also detected at the protein level, indicating that the transfection system accurately and effectively overexpressed porcine primary myoblasts. Figure 5 Shown are the results of MyoG gene expression after overexpression of two MyoG plasmids in porcine primary myoblasts.

[0040] At the same time, the changes in the expression levels of MyoD and MyHC genes were detected. Both MyoD and MyHC genes play an important regulatory role in myogenic differentiation. The mRNA and protein expressions of MyoD and MyHC showed that both were more highly expressed in primary myoblasts transfected with the MyoG plasmid of Western lean pigs. Figure 6 Shown are the expression results of MyoD and MyHC after overexpression of two MyoG plasmids in primary porcine myoblasts.

[0041] Example 2: SNP rs3471653254C>T mutation affects the enrichment level of transcription factor HOXA5 in the promoter region, thereby regulating the transcriptional expression of LncRNAmyoparr and MyoG

[0042] After overexpressing two MyoG vectors, cells were treated with 1% formaldehyde to crosslink proteins with DNA. After cell lysis, ultrasonic fragmentation was used to form small chromatin fragments, and antigen-antibody reaction was used to promote immunoprecipitation. Subsequently, NaCl and proteinase K were used to remove DNA-protein crosslinks and then DNA was purified and recovered. Finally, 1.8% agarose gel electrophoresis and RT-PCR were used for further analysis of DNA.

[0043] Figure 7The results of ChIP-qPCR analysis of the enrichment of HOXA5 in the promoter region of the MyoG gene in two major pig breeds are shown. Through the ChIP-qPCR experiment, it can be seen that the transcription factor HOXA5 has a higher enrichment level in the promoter region of Western lean pigs.

[0044] The two promoter fragments before and after the SNP rs3471653254 mutation were cloned and inserted into the XhoI / NotI site of psiCHECK to construct a vector to obtain a dual-luciferase plasmid containing the target fragment. The reporter gene plasmid and the HOXA5 overexpression plasmid were co-transfected into cells. It can be seen that when HOXA5 is overexpressed, the fluorescence activity of the promoter region is significantly increased after the SNP rs3471653254 site is converted from C to T, indicating that this site plays an important role in regulating transcription. Figure 8 The results of analyzing the fluorescence activity of the promoter fragments with the SNP rs3471653254 site being a T base or a C base respectively using the dual fluorescence reporter system are shown.

[0045] Example 3: LncRNAmyoparr interacts with miR-30b-3p

[0046] The predicted binding site of LncRNAmyoparr with miR-30b-3p was mutated, and wild-type and mutant LncRNAmyoparr fragments were designed respectively ( Fig. 9 , A), and cloned into the expression vector of firefly luciferase. It can be seen that when miR-30b-3p is overexpressed, the expression of miR-30b-3p inhibits the fluorescence activity intensity of wild-type LncRNAmyoparr, but when the binding site is mutated, miR-30b-3p cannot bind to it, resulting in no significant change in the fluorescence value. This confirms that the two bind through the predicted site. Fig. 9 As can be seen in B, the expression trends of the two are antagonistic. Fig. 9 As shown, the dual fluorescence reporter system was used to analyze the interaction between LncRNAmyoparr and miR-30b-3p through the predicted binding sites and the expression trend analysis of the two.

[0047] Example 4: miR-30b-3p inhibits myogenesis in primary porcine myoblasts

[0048] After treating porcine primary myoblasts with a specific miR-30b-3p agonist (custom agomir agonist, Guangzhou Ruibo Biotechnology), the efficiency of cell differentiation into myoblasts was greatly reduced. Conversely, after treating with a miR-30b-3p inhibitor (custom antagomir inhibitor, Guangzhou Ruibo Biotechnology), the level of cell myogenic differentiation was significantly improved. It can be seen that miR-30b-3p has a regulatory effect of inhibiting myogenesis during myogenesis. Fig.10 Shown are the results of differentiation of porcine primary myoblasts treated with agonists and inhibitors of miR-30b-3p.

[0049] The present invention is not limited to the above-mentioned embodiments. If various changes or modifications to the present invention do not depart from the spirit and scope of the present invention, and if these changes and modifications fall within the scope of the claims and equivalent technologies of the present invention, the present invention is also intended to include these changes and modifications.

Claims

1. Application of the SNP mutation site of the pig MyoG gene in breeding for improving pig lean meat yield performance, characterized in that: The SNP mutation site is the SNPrs3471653254 C>T mutation located at the upstream promoter of the pig MyoG gene and 299 bp before the transcription start site.

2. Application of a reagent for detecting the SNP mutation site of the pig MyoG gene in breeding for improving pig lean meat yield performance, characterized in that: The SNP mutation site is the SNPrs3471653254 C>T mutation located at the upstream promoter of the pig MyoG gene and 299 bp before the transcription start site.

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