Use of parp3 gene in regulating development of porcine placenta and method

Abstract

The application discloses PARP3 Application and method of gene in regulation of pig placenta development, and relates to the field of animal genetics. PARP3 The gene is low in methylation level and high in expression in the HBW placenta. PARP3 Therefore, the methylation level or expression level of the gene can be used for regulating the quality of pig placenta development, further improving the birth weight and survival rate of piglets, and further improving the reproductive efficiency of sows and the market competitiveness of pig farms.

Description

Technical Field

[0001] This invention relates to the field of gene regulation technology, and particularly to a... PARP3 Applications and methods of gene regulation in porcine placental development. Background Technology

[0002] The reproductive performance of sows can be assessed using various indicators, among which the birth weight of piglets plays a crucial role and is also a key indicator affecting economic efficiency in actual pig production. Piglets with low birth weights not only have a significantly increased risk of morbidity and mortality before weaning, but also negatively impact their later growth, specifically manifesting as decreased feed utilization efficiency and weakened growth performance. These problems ultimately lead to serious economic losses in pig production.

[0003] The placenta is the organ through which substances are exchanged between the sow and fetus during pregnancy, playing a crucial role in the growth and development of piglets within the womb. For example, the placenta transports essential nutrients such as oxygen, glucose, and amino acids to the fetus, while metabolic waste products such as urea and carbon dioxide produced by the fetus are also transported to the mother and excreted via the placenta. The porcine placenta develops rapidly during pregnancy. The trophoblast cells (PTr2 cells) are the most important functional cells in the placenta, beginning differentiation as early as the morula stage. They not only directly mediate embryo implantation but also form the core of subsequent placental structural and functional development. After successful pregnancy, trophoblast cells (PTr2 cells) synthesize and secrete various essential bioactive substances and nutrients, providing crucial support for fetal growth and development. As the core cells of the placenta, the migration, invasion, and proliferation capabilities of trophoblast cells are the cellular essence of placental development. The orderly expression and temporal control of these three aspects are essential conditions for placental formation, establishment of maternal-fetal connection, and the perfecting of placental function.

[0004] The PARP family plays a central role in DNA repair, genome stability maintenance, RNA interference, and transcriptional regulation. Among the 17 genes in the PARP family, the following genes play particularly important roles: PARP1 , PARP2 and PARP3 . PARP3 and PARP1 , PARP2 They share similar protein homology structures, containing three functional domains: an N-terminal specific domain, a central WGR domain, and a C-terminal catalytic domain containing a helical region. However... PARP3 There are no reports on whether genes are related to placental development. Summary of the Invention

[0005] The purpose of this invention is to provide aPARP3 The application and methods of gene regulation in porcine placental development aim to improve the developmental quality of porcine placenta and further increase the birth weight of piglets.

[0006] According to a first aspect of the invention, a method is provided PARP3 Application of genes in regulating porcine placental development. Therefore, through regulation... PARP3 The level of gene expression can improve the development quality of the pig placenta, further increase the birth weight of piglets, and improve the reproductive efficiency and market economic value of sows.

[0007] In some implementations, the application is achieved by regulating... PARP3 This is achieved through the level of gene expression.

[0008] In some implementations, the application is achieved by regulating... PARP3 This is achieved through the level of gene methylation.

[0009] According to a second aspect of the invention, a method is provided PARP3 The application of genes in the preparation of products for regulating porcine placental development. This will allow for the regulation of... PARP3 The reagents used to measure gene expression levels can be used to improve the development quality of the pig placenta, further increase the birth weight of piglets, and improve the reproductive efficiency and market value of sows.

[0010] In some implementations, the application is achieved by regulating... PARP3 This is achieved through the level of gene expression.

[0011] In some implementations, the application is achieved by regulating... PARP3 This is achieved through the level of gene methylation.

[0012] According to a third aspect of the invention, a method is provided PARP3 Non-therapeutic applications of genes in promoting the proliferation and / or migration of porcine placental trophoblast cells. Thus, through regulation... PARP3 The level of gene expression can promote the proliferation and / or migration of trophoblast cells in the porcine placenta, thereby improving the developmental quality of the sow's placenta, further increasing the birth weight of piglets, and enhancing the reproductive efficiency and market economic value of sows.

[0013] In some implementations, the application is achieved by regulating... PARP3 This is achieved through the level of gene expression.

[0014] In some implementations, the application is achieved by regulating... PARP3 This is achieved through the level of gene methylation.

[0015] According to a fourth aspect of the invention, a method is provided. PARP3Application of genes in the preparation of products that promote the proliferation and / or migration of porcine placental trophoblast cells. This will regulate... PARP3 The reagents used to produce products that enhance gene expression levels can be used to promote the proliferation and / or migration of trophoblast cells in the porcine placenta, thereby improving the developmental quality of the sow placenta, further increasing the birth weight of piglets, and enhancing the reproductive efficiency and market economic value of sows.

[0016] In some implementations, the application is achieved by regulating... PARP3 This is achieved through the level of gene expression.

[0017] In some implementations, the application is achieved by regulating... PARP3 This is achieved through the level of gene methylation.

[0018] According to a fifth aspect of the invention, a non-therapeutic method for improving the quality of placental development in pigs is provided, the method comprising adding a substance to the sow's feed or drinking water that can promote placental development. PARP3 Agents that can inhibit gene expression may PARP3 Reagents for regulating gene methylation levels. Therefore, this method can efficiently and conveniently regulate... PARP3 Improving gene expression levels can enhance the developmental quality of the sow placenta, thereby increasing piglet birth weight, sow reproductive efficiency, and market economic value.

[0019] According to a sixth aspect of the invention, a non-therapeutic method for improving the quality of porcine placental development is provided, the method comprising directly injecting into the placenta a substance that can promote… PARP3 Agents that can inhibit gene expression may PARP3 Reagents for regulating gene methylation levels. Therefore, this method can efficiently and conveniently regulate... PARP3 Improving gene expression levels can enhance the developmental quality of the sow placenta, thereby increasing piglet birth weight, sow reproductive efficiency, and market economic value.

[0020] In some implementations, the promotion PARP3 Reagents for gene expression include those that can overexpress gene. PARP3 An overexpression plasmid of the gene, the nucleotide sequence of which is shown in SEQ ID No:5.

[0021] In some implementations, the above can suppress PARP3 The reagent used to measure gene methylation levels was decitabine.

[0022] According to a seventh aspect of the invention, a non-therapeutic method for increasing birth weight in pigs is provided, said method being achieved by improving the quality of placental development in pigs, said method comprising adding a substance to the sow's feed or drinking water that can promote... PARP3 Agents that can inhibit gene expression mayPARP3 Reagents for regulating gene methylation levels. Therefore, this method can efficiently and conveniently regulate... PARP3 Improving gene expression levels can increase piglet birth weight, thereby further improving piglet survival rate, sow reproductive efficiency, and market economic value.

[0023] According to an eighth aspect of the invention, a non-therapeutic method for increasing birth weight in pigs is provided, said method being achieved by improving the quality of placental development in pigs, said method involving direct injection into the placenta to promote... PARP3 Agents that can inhibit gene expression may PARP3 Reagents for regulating gene methylation levels. Therefore, this method can efficiently and conveniently regulate... PARP3 Improving gene expression levels can increase piglet birth weight, thereby further improving piglet survival rate, sow reproductive efficiency, and market economic value.

[0024] In some implementations, the promotion PARP3 Reagents for gene expression include those that can overexpress gene. PARP3 An overexpression plasmid of the gene, the nucleotide sequence of which is shown in SEQ ID No:5.

[0025] In some implementations, the above can suppress PARP3 The reagent used to measure gene methylation levels was decitabine.

[0026] According to a ninth aspect of the present invention, a non-therapeutic method for promoting the proliferation and / or migration of porcine placental trophoblast cells is provided, the method comprising injecting or transfecting porcine placental trophoblast cells with a promoter PARP3 Agents that can inhibit gene expression may PARP3 A reagent for gene methylation levels. Therefore, this method can promote the proliferation and / or migration of porcine placental trophoblast cells, thereby improving the developmental quality of the sow placenta, further increasing piglet birth weight, and enhancing sow reproductive efficiency and market economic value.

[0027] In some implementations, the promotion PARP3 Gene expression reagents include those that can overexpress gene. PARP3 An overexpression plasmid of the gene, the nucleotide sequence of which is shown in SEQ ID No:5.

[0028] In some implementations, the above can suppress PARP3 Decitabine is a reagent for measuring gene methylation levels.

[0029] According to a tenth aspect of the present invention, a product is provided that can promote the development of the porcine placenta, said product containing ingredients that can promote… PARP3 Agents that can inhibit gene expression may PARP3A reagent for increasing gene methylation levels. Therefore, this product can promote placental development in pigs, thereby improving the reproductive efficiency and market value of sows.

[0030] In some implementations, the promotion PARP3 Reagents for gene expression include those that can overexpress gene. PARP3 An overexpression plasmid of the gene, the nucleotide sequence of which is shown in SEQ ID No:5.

[0031] In some implementations, the above can suppress PARP3 The reagent used to measure gene methylation levels was decitabine.

[0032] According to the eleventh aspect of the invention, a method for promoting PARP3 The application of gene expression reagents in the preparation of products that promote porcine placental development. Therefore, by promoting... PARP3 Products made from gene expression reagents can promote placental development in pigs, thereby improving the reproductive efficiency and market value of sows.

[0033] In some implementations, the promotion PARP3 Reagents for gene expression include those that can overexpress gene. PARP3 An overexpression plasmid of the gene, the nucleotide sequence of which is shown in SEQ ID No:5.

[0034] In some implementations, the promotion PARP3 Agents for gene expression include those that can inhibit PARP3 A reagent for gene methylation levels, wherein the reagent is decitabine.

[0035] According to the twelfth aspect of the invention, a method for promoting PARP3 The application of gene expression reagents in the preparation of products that promote the proliferation and / or migration of porcine placental trophoblast cells. Thus, by promoting... PARP3 The gene expression reagents can be made into products that can promote the proliferation and / or migration of trophoblast cells in the porcine placenta, thereby improving the developmental quality of the sow placenta and further increasing the birth weight of piglets, thus improving the reproductive efficiency and market economic value of sows.

[0036] In some implementations, the promotion PARP3 Reagents for gene expression include those that can overexpress gene. PARP3 An overexpression plasmid of the gene, the nucleotide sequence of which is shown in SEQ ID No:5.

[0037] In some implementations, the promotion PARP3 Agents for gene expression include those that can inhibit PARP3 A reagent for gene methylation levels, wherein the reagent is decitabine.

[0038] According to the thirteenth aspect of the invention, a method for promoting PARP3 The application of gene expression reagents in the preparation of products that increase piglet birth weight. Therefore, by promoting... PARP3 Products made from gene expression reagents can improve the quality of placental development in pigs, thereby increasing the birth weight of piglets.

[0039] In some implementations, the promotion PARP3 Reagents for gene expression include those that can overexpress gene. PARP3 An overexpression plasmid of the gene, the nucleotide sequence of which is shown in SEQ ID No:5.

[0040] In some implementations, the promotion PARP3 Agents for gene expression include those that can inhibit PARP3 A reagent for gene methylation levels, wherein the reagent is decitabine.

[0041] According to the fourteenth aspect of the present invention, a regulation is provided. PARP3 The application of gene expression reagents in the preparation of products that regulate the PI3K-Akt pathway in porcine placental trophoblast cells. Thus, by regulating... PARP3 The product made from the gene expression reagent can regulate the PI3K-Akt signaling pathway in porcine placental trophoblast cells and further regulate the proliferation or apoptosis of porcine placental trophoblast cells to maintain placental homeostasis and development, thereby affecting the birth weight of piglets.

[0042] The beneficial effects of this invention are: This study collected placental samples from four litters of the same sex, specifically high birth weight (HBW) and low birth weight (LBW) Large White piglets. RNA-seq and WGBS sequencing were used to reveal differentially modified DNA methylation genes in the placentas of HBW and LBW litters. PARP3 To investigate the differential regulation and significant differential expression of DNA methylation. PARP3 Effects on the development of placental trophoblast cells (PTr2 cells). By studying the methylation level of this gene, it was found that... PARP3 The methylation level of the gene in LBW placenta was significantly higher than that in HBW placenta, suggesting that PARP3 Gene methylation status may be closely related to placental development and fetal growth regulation. Then, it regulates PTr2 cells through DNA methylation. PARP3 The expression of [a specific substance] was investigated, and the results showed that the DNA methylation inhibitor 5-Aza could induce [a specific effect] in PTr2 cells. PARP3 Gene expression. This further confirms that during placental development, PARP3Gene expression is regulated by DNA methylation, and in PTr2 cells treated with 5-Aza... PARP3 Reduced methylation levels in promoter regions and cells PARP3 Expression was upregulated. This was then achieved by either inhibiting or overexpressing PTr2 cells. PARP3 Gene, results showed overexpression PARP3 Genes can promote PTr2 cell proliferation and migration. PTr2 cells play an important role in placental development; promoting PTr2 cell proliferation and migration can effectively promote placental development and further increase piglet birth weight. Furthermore, inhibiting PTr2 cell proliferation and migration can also contribute to placental development. PARP3 Gene expression significantly inhibits the PI3K-Akt signaling pathway, downregulating PI3K and mTOR gene expression. This indicates regulation... PARP3 Gene expression can also regulate the PI3K-Akt signaling pathway in the placenta to maintain placental homeostasis and development, thereby affecting the birth weight of piglets. Therefore, it is possible to regulate... PARP3 Gene methylation levels or expression levels can regulate the quality of porcine placental development and further improve piglet birth weight; it can also be achieved by regulating... PARP3 Gene methylation levels or expression levels can promote the proliferation and / or migration of porcine placental trophoblast cells; they can also be regulated by... PARP3 Gene methylation levels or expression levels regulate the PI3K-Akt signaling pathway in porcine placental trophoblast cells, thereby regulating the proliferation or apoptosis of placental trophoblast cells to maintain placental homeostasis and development, thus affecting the birth weight of piglets. Therefore, by regulating... PARP3 The level of gene methylation or expression can be used to improve the quality of pig placental development, further increase the birth weight and survival rate of piglets, and thus improve the reproductive efficiency of sows and the market competitiveness of breeding farms. Attached Figure Description

[0043] Figure 1 Four-quadrant scatter plot for combined analysis of RNA-seq and WGBS sequencing; Figure 2 For joint analysis of Venn diagrams; Figure 3 For DEG-DMG GO enrichment analysis; Figure 4 DEG-DMG KEGG enrichment analysis plot; Figure 5 For placenta PARP3 methylation status diagram of gene promoter regions; Figure 6 For RT-qPCR validation PARP3 Map showing mRNA expression levels in placental tissue; Figure 7After treatment of PTr2 cells with decitabine PARP3 Image showing the results of methylation status detection in gene promoter regions; Figure 8 This is a graph showing the expression of various genes in PTr2 cells after treatment with decitabine. Figure 9 Interference with RT-qPCR detection PARP3 The results of mRNA expression detection of post-proliferation and apoptosis marker genes; Figure 10 For interference PARP3 Experimental results on gene-induced proliferation of PTr2 cells: Among them, Figure 10 In this context, 'a' indicates CCK-8 detection interference. PARP3 Results of post-cell proliferation assay; Figure 10 In this context, 'b' represents EdU detection interference. PARP3 Results of post-cell proliferation assay; Figure 11 For interference PARP3 Scratch assay results of PTr2 cells with gene inhibition of migration: scale bar is 500 μm; Figure 12 For RT-qPCR detection of overexpression PARP3 The mRNA expression results of post-proliferation and apoptosis marker genes; Figure 13 For overexpression PARP3 Figure showing the experimental results of gene-induced proliferation of PTr2 cells: (The figure is missing from the original text.) Figure 13 In this context, 'a' indicates that CCK-8 is used to detect overexpression. PARP3 Post-cell proliferation results; Figure 13 In this context, 'b' indicates that EdU is used to detect overexpression. PARP3 Post-cell proliferation results; Figure 14 For overexpression PARP3 Figure 1: Results of scratch assay for gene-induced migration of PTr2 cells: scale bar is 500 μm. Figure 15 Volcano plot of differentially expressed genes in the cell transcriptome; Figure 16 A graph showing GO enrichment analysis of the cellular transcriptome; Figure 17 This is a graph showing the KEGG enrichment analysis of the cellular transcriptome. Figure 18 The results of the detection of key marker genes in the PI3K-Akt signaling pathway for RT-qPCR validation are shown in the figure. Detailed Implementation

[0044] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various changes and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

[0045] Unless otherwise specified, all chemical substances, proteins, enzymes, or reagent kits used in this invention are commercially available.

[0046] Unless otherwise stated, the methods used in this invention, such as PCR amplification and nucleic acid extraction, all employ conventional experimental procedures in the field. Related operations can be performed with reference to commonly used experimental technical manuals or the operating instructions for commercial reagent kits and instruments.

[0047] Example 1: Differentially expressed genes modified by differential methylation were screened from pig HBW and LBW placentas.

[0048] 1.1 Sample collection and data analysis.

[0049] Piglets and placentas expelled by sows during farrowing were collected, and the placentas and their associated piglets were weighed. The condition of the piglets and umbilical cords was recorded. Four pairs of placentas from litters of the same sex, belonging to both high birth weight (HBW) and low birth weight (LBW), were selected for the experiment. It was found that the placentas of HBW piglets were larger and thicker with abundant blood vessels; the placentas of LBW piglets were smaller and narrower with sparse blood vessels, and the placentas of HBW piglets were consistently heavier than those of LBW piglets (Table 1).

[0050] Table 1 Information on sequenced piglets and sows 4-31 HBW1 Dam 1368 g 250 g 4-31 LBW1 Dam 646 g 107 g 7-1 HBW2 Sire 909 g 252 g 7-1 LBW2 Sire 415 g 174.4 g 7-3 HBW3 Dam 1200 g 235.6 g 7-3 LBW3 Dam 772 g 132.8 g 11-3 HBW4 Dam 1400 g 241.3 g 11-3 LBW4 Dam 500 g 101.6 g Transcriptome sequencing (RNA-seq) and whole-genome methylation sequencing (WGBS) were performed on placental samples from HBW and LBW piglets, and the data quality was assessed. Data summaries are shown in Tables 2 and 3. (1) RNA-seq sequencing data (Table 2): Transcriptome data of each group of placental samples yielded more than 34 million clean reads, of which the number of reads aligned to the reference genome was around 30 million. The alignment rate of the eight samples ranged from 79.5% to 84.4%, indicating that the data quality was high and met the requirements for subsequent bioinformatics analysis; (2) WGBS sequencing data (Table 3): The average alignment rate between the sequencing data and the reference genome was 62.275%, and the BS conversion rate was excellent, with an average value exceeding 99.57%. The above indicators show that the whole-genome methylation sequencing data in this study have both good integrity and high accuracy, and can provide reliable data support for subsequent related analyses.

[0051] Quality assessment of the sequencing data confirmed that the RNA-seq and WGBS data of the HBW and LBW piglet placental samples met the analytical requirements, indicating that the sequencing data of all samples were of reliable quality and suitable for subsequent analysis.

[0052] Table 2. Quality control and alignment of transcriptome data

[0053] Table 3 WGBS Data Quality Control and Comparison Status HBW1 336285596 207210011 61.6 99.56 LBW1 356490955 222656647 62.5 99.50 HBW2 337569551 213872280 63.4 99.59 LBW2 373831535 233669886 62.5 99.57 HBW3 311402550 194120465 62.3 99.59 LBW3 283050797 176140333 62.2 99.55 HBW4 337256193 208664569 61.9 99.59 LBW4 275433627 170224645 61.8 99.55 Analysis results combining whole-genome methylation and transcriptome sequencing data, such as Figure 1 As shown: This indicates that the genes with high expression of hypomethylation include WNT2B Genes with high methylation and low expression include HBEGF , PARP3 The joint analysis results are as follows: Figure 2 As shown: 68 DEGs whose promoter regions are regulated by DNA methylation were identified. The GO enrichment analysis results of the combined analysis are as follows... Figure 3 As shown, differentially expressed methylated genes are mainly enriched in processes such as ribonucleoside biosynthesis, ribonucleoside metabolism, glycosyl compound biosynthesis, and pentosyltransferase activity. The combined KEGG enrichment analysis results are as follows: Figure 4 As shown, differentially expressed methylated genes are mainly enriched in pathways such as calcium signaling, the phosphatidylinositol signaling system, glycosaminoglycan biosynthesis-keratin sulfate, and arginine and proline metabolism. Finally, by combining gene expression fold-over with differential methylation fold-over, the most suitable pathways were selected. PARP3 Further research is needed on the function and mechanism of genes.

[0054] Example 2 PARP3 Tissue-level expression and DNA methylation levels.

[0055] 2.1 PARP3 Gene promoter region sequence analysis.

[0056] The analysis was performed using MethPrimer software. PARP3 The sequence of the gene promoter region was determined, with parameters set to G+C content > 50% and CpG observed / expected ratio > 0.6. Results showed that:PARP3 The promoter region contains two CpG islands and multiple CpG sites. For further analysis, a [design / design / engineering method] was developed. PARP3 The M (methylation) specific primers and U (unmethylation) specific primers for the promoter region of the gene are shown in Table 4.

[0057] Table 4. Methylation and Non-methylation Specific Primers

[0058] 2.2 At the organizational level PARP3 DNA methylation in the gene promoter region.

[0059] Genomic DNA was extracted from placental tissues of HBW and LBW individuals. Quality testing confirmed that its concentration and purity met the requirements for subsequent experiments. Using this DNA as a template, after bisulfite conversion, methylation-specific PCR (MS-PCR) was employed to... PARP3 The methylation status of the gene promoter region was detected.

[0060] MS-PCR detection method: (1) Using MethPrimer for web page design PARP3 The methylated-specific and unmethylated-specific primers for the CpG sites in the promoter region are shown in Table 4.

[0061] (2) Using bisulfite-converted DNA as a template, MS-PCR amplification was performed using 2×Taq PCR Star Mix. The reaction system is shown in Table 5, and the reaction program is shown in Table 6.

[0062] Table 5 MS-PCR reaction system

[0063] Table 6 MS-PCR Reaction Temperature 95 ℃ 5 min 95 ℃ 30 sec 40 cycles 56 ℃ 30 sec 40 cycles 72 ℃ 30 sec 40 cycles 72 ℃ 5 min 16 ℃ 2 min

[0064] (3) After PCR amplification, mix 5 μL of the amplification product with 1 μL of 6× DNA Loading Buffer. Use a 2.0% agarose gel, add the DNA marker and sample sequentially, and perform electrophoresis at a constant voltage of 150V. Observe the DNA bands using an imaging system after the amplification is complete.

[0065] (4) Based on the agarose gel electrophoresis results, the methylation status can be determined according to the band pattern: Unmethylated: Only unmethylated bands were detected.

[0066] Fully methylated: Only methylated bands were detected.

[0067] Partial methylation: Both methylated and unmethylated bands were detected.

[0068] (5) The degree of methylation can be quantified by the following formula: calculate the percentage of the gray value of the methylated band to the sum of the gray values ​​of the methylated and non-methylated bands, that is, the degree of methylation (%) = [gray value of methylated band / (gray value of methylated band + gray value of non-methylated band)] × 100%.

[0069] Test results as follows Figure 5 As shown, the results indicate that in LBW placenta, PARP3 The DNA methylation level in the gene promoter region was significantly higher than that in the HBW placenta, indicating that... PARP3 The methylation status of genes may be closely related to placental development and fetal growth regulation.

[0070] 2.3 PARP3 Gene expression in placental tissue.

[0071] RNA was extracted from placental tissues of HBW and LBW patients. Quality testing confirmed that the concentration and purity met the requirements for subsequent experiments. Using this RNA as a template, cDNA was reverse transcribed and then subjected to RT-qPCR. The results are as follows: Figure 6 As shown, the results indicate that in LBW placenta PARP3 The expression level of mRNA was significantly lower than that in HBW placenta. P <0.05).

[0072] 2.4 At the cellular level PARP3 DNA methylation in the gene promoter region.

[0073] Genomic DNA was extracted from PTr2 cells treated with 5 μM decitabine (5-Aza) (a methylation inhibitor) and DMSO (control group). Quality testing confirmed that the concentration and purity met the requirements for subsequent experiments. Using this DNA as a template, after bisulfite conversion, methylation-specific PCR (MS-PCR) was performed to... PARP3 The methylation status of the gene promoter region was detected. Results are as follows: Figure 7 As shown, the results indicate that in the DMSO control group, PARP3 The DNA methylation level in the gene promoter region was higher than that in the decitabine-treated group, demonstrating that DNA methylation occurs during placental development. PARP3 Gene expression is regulated by DNA methylation, and in PTr2 cells treated with 5-Aza... PARP3The methylation level in the promoter region was decreased. RT-qPCR was performed on randomly selected differentially expressed genes regulated by DNA methylation, and the results are as follows: Figure 8 As shown: This indicates that treatment with 5 μM decitabine not only... PARP3 Gene expression levels were significantly higher than those in the DMSO control group, and the expression levels of other differentially expressed methylated gene mRNAs were also significantly higher than those in the control group.

[0074] Example 3 PARP3 Effects on PTr2 cell function.

[0075] 3.1 Synthesis of PARP3 overexpression vector pcDNA3.1-PARP3.

[0076] The pigs used in this study PARP3 The gene overexpression vector was synthesized by Beijing Qingke Biotechnology Co., Ltd. The specific construction process is as follows: using pigs... PARP3 Gene( Ensembl:ENSSSCG00000023033 Using the coding sequence (CDS) of the target gene as a template, specific primers were designed and PCR amplification was performed to obtain the target fragment. This fragment was then cloned into the multiple cloning site of the eukaryotic expression vector pcDNA3.1 (Beijing Qingke Biotechnology Co., Ltd., GZ0289627-1) to construct the target gene. PARP3

[0077] 3.2 PARP3 Synthesis of interference fragments.

[0078] PARP3 The interfering fragment was designed and synthesized by Suzhou Genecasting Co., Ltd., and the interfering sequence is shown in Table 7.

[0079] Table 7 PARP3 Interference sequence

[0080] 3.3 Interference PARP3 Effects on Ptr cell proliferation and apoptosis genes.

[0081] si-NC and si- PARP3 Transfected into PTr2 cells (porcine placental trophoblast cells), then interference was detected. PARP3 The expression levels of proliferation and apoptosis genes in PTr2 cells, and the primer sequences for RT-qPCR detection of each gene are shown in Table 8. The detection results are as follows: Figure 9 As shown, the results indicate that interference suppression PARP3 After expressing, PARP3 The mRNA expression level was significantly downregulated ( Figure 9 a) indicates that si- PARP3 The transfection and interference suppression effects are very good. Meanwhile, as... Figure 9 The results in b show: interference suppression PARP3 After expression, proliferation marker genes in PTr2 cells KI67 The mRNA level was significantly reduced, and the anti-apoptotic gene was also significantly reduced. BCL2 The mRNA level was significantly reduced, and apoptosis marker genes were also significantly reduced. CASP3 , CASP9 The mRNA expression level was significantly increased. These results indicate that interference inhibits... PARP3 Expression of this substance promoted apoptosis of PTr2 cells and inhibited their proliferation.

[0082] Table 8 qPCR primer sequences

[0083] 3.4 Interference PARP3 Inhibits the proliferation of PTr2 cells.

[0084] si-NC and si- PARP3 The cells were transfected into PTr2 cells, and CCK8 and Edu assays were performed to detect their proliferation capacity.

[0085] CCK-8 cell proliferation detection method: (1) Cell culture and treatment: Cell proliferation activity was detected at 24, 48 and 72 h after transfection; (2) CCK-8 detection: Add 10 μL of CCK-8 solution to each well of the 96-well plate and incubate in an incubator for 2 h; (3) OD value determination: The optical density (OD) of each well at 450 nm was determined using an ELISA reader.

[0086] EdU method for cell proliferation detection: (1) 48 hours after transfection, the cells were treated with culture medium containing EdU solution and incubated in a 37 ℃, 5% CO2 incubator for 2 hours.

[0087] (2) Remove the culture medium with a pipette and wash the cells twice with DPBS for 3 minutes each time. Then add 4% paraformaldehyde and fix the cells at room temperature for 15 minutes. After fixation, wash twice more with DPBS. Next, treat the cells with permeabilization buffer for 10 minutes, and then wash twice with DPBS to remove the permeabilization buffer.

[0088] (3) Add 70 μL of freshly prepared Click reaction mixture to each well and incubate at room temperature for 30 minutes. After the reaction, aspirate the reaction solution and wash twice with DPBS. Then add DAPI staining solution for nuclear counterstaining and incubate at room temperature for 8 minutes. Finally, aspirate the staining solution and complete the final wash with DPBS.

[0089] (4) Use a fluorescence microscope to acquire images. Randomly select 6 fields of view for each well to take pictures and perform statistical analysis on the image data.

[0090] CCK8 test results are as follows Figure 10 As shown in Figure a, the results indicate that interference PARP3 Subsequently, the proliferation of PTr2 cells was significantly inhibited.

[0091] EdU experimental results are as follows Figure 10 As shown in Figure b, the results indicate that interference PARP3 Subsequently, the proliferation of PTr2 cells was significantly inhibited.

[0092] In summary, interference PARP3 Afterwards, it can significantly inhibit the proliferation of PTr2 cells.

[0093] 3.5 Interference PARP3 Inhibits the migration of Ptr cells.

[0094] To clarify PARP3 To investigate the role of genes in the migration of PTr2 cells, si-PARP3 and the negative control si-NC were transfected into PTr2 cells, and the differences in migration ability between the two groups were observed and compared by cell scratch assay.

[0095] Scratch assay for cell migration: (1) Cell seeding and scratching: PTr2 cells were seeded in 6-well plates. After 36 hours of transfection to form a monolayer, the cells were scratched with a 200 μL pipette tip, washed with DPBS, and then cultured in serum-free medium for 12 hours.

[0096] (2) Image acquisition and analysis: The wound closure status was observed and photographed under a microscope. Three fields of view were randomly selected, and the wound area at 0h and 12h was calculated using ImageJ software. The migration rate was calculated according to the formula: Migration rate = (0h wound area – 12h wound area) / 0h wound area × 100%.

[0097] The results are as follows Figure 11 As shown, the results indicate that PTr2 cells in the interference group (transfected with si-PARP3) migrated significantly slower and farther within the same time frame. Therefore, interference... PARP3 It can significantly inhibit the migration ability of PTr2 cells.

[0098] 3.6 Overexpression PARP3 Effects on PTr2 cell proliferation and apoptosis genes.

[0099] In order to study overexpression PARP3 The role of genes in the proliferation and apoptosis gene expression of PTr2 cells, transfection PARP3 After the overexpression vector pcDNA3.1-PARP3 and the empty vector pcDNA3.1 were introduced into PTr2 cells, the results were as follows: Figure 12 As shown, the results indicate that overexpression PARP3 Afterwards, proliferation marker genes PCNA The mRNA level was significantly increased, and the anti-apoptotic gene was also present. BCL2 The mRNA level was significantly elevated, and apoptosis marker genes were also observed. CASP9 The mRNA expression level was significantly reduced. These results indicate that overexpression... PARP3 The gene promoted the proliferation of PTr2 cells and inhibited their apoptosis.

[0100] 3.7 Overexpression PARP3 Promotes the proliferation of PTr2 cells.

[0101] In order to study overexpression PARP3 The effect of genes on PTr2 cell proliferation, transfection PARP3 The overexpression vector pcDNA3.1-PARP3 and the empty vector pcDNA3.1 were introduced into PTr2 cells, and then CCK8 and EdU assays were performed. The results are as follows. Figure 13 As shown: CCK8 results (Figure 13 a) indicates that overexpression PARP3 Significantly promoted the proliferation of PTr2 cells; EdU results ( Figure 13 b) indicates that overexpression PARP3 It significantly promoted the proliferation of PTr2 cells. In summary, overexpression... PARP3 After gene administration, the proliferation of PTr2 cells can be significantly promoted.

[0102] 3.8 Overexpression PARP3 Promotes the migration of Ptr cells.

[0103] To further study overexpression PARP3 The role of genes in PTr2 cell migration, specifically in the migration of pcDNA3.1- PARP3 PTr2 cells were transfected with the empty vector pcDNA3.1, and the difference in migration ability between the two groups of cells was observed and compared by cell scratch assay. Results are as follows: Figure 14 As shown in the figure, the results indicate that PTr2 cells in the overexpression group migrated significantly faster and farther within the same time frame. In conclusion, overexpression... PARP3 Genes can promote the migration ability of PTr2 cells.

[0104] Example 4 PARP3 Effects on the PI3K-Akt signaling pathway in PTr2 cells.

[0105] In order to investigate PARP3 The mechanism regulating PTr cells, transfecting PTr2 cells with si- PARP3 Interfering fragments were extracted, with si-NC as a control group. Cellular RNA was then extracted and sent for transcriptome sequencing. The results of the differentially expressed gene volcano plot screening in the cell transcriptome are shown below. Figure 15 As shown, the results indicate that a total of 528 upregulated DEGs and 802 downregulated DEGs were identified. Among the upregulated DEGs, the one with the most significant p-value was... SERPINB2 , BAIAP2L1 The most significant reduction in DEGs p-values ​​was... ADGRD1 , IGFBP5 The results of GO enrichment analysis on the screened DGEs are as follows: Figure 16 As shown in the figure, the analysis indicates that DGEs are significantly enriched in biological functions such as enzyme-linked receptor protein signaling pathway, cell adhesion, stem cell differentiation, transmembrane receptor protein tyrosine kinase signaling pathway, tube morphogenesis, and salt transmembrane transport activity. KEGG enrichment analysis results are shown below. Figure 17 As shown, DGEs are significantly enriched in the complement and coagulation cascade pathway, the PI3K-Akt signaling pathway, etc.

[0106] The extracted RNA was analyzed by RT-qPCR. The primer sequences are shown in Table 9. The results are as follows: Figure 18 As shown, the results indicate that interference in PTr2 cells... PARP3 After expression, key molecules in the PI3K-Akt signaling pathway PI3K and its important downstream regulatory factors mTOR The expression levels of these substances were significantly reduced, indicating that inhibition was achieved in PTr2 cells. PARP3 Expression can significantly inhibit the PI3K-Akt signaling pathway and downregulate it. PI3K and mTOR The expression. This illustrates that through regulation PARP3 Gene expression can regulate the PI3K-Akt signaling pathway in porcine placental trophoblast cells and further regulate the proliferation or apoptosis of porcine placental trophoblast cells to maintain placental homeostasis and development, thereby affecting the birth weight of piglets.

[0107] Table 9 Primers for detecting marker genes in the PI3K-Akt signaling pathway

[0108] In summary, this study used sex-matched Large White pigs from the same litter, collecting placental tissues from both high birth weight (HBW) and low birth weight (LBW) individuals. RNA-seq and whole-genome bisulfite sequencing (WGBS) were combined to systematically analyze genes differentially regulated by DNA methylation in the placenta. The results showed that... PARP3 This is a key gene that simultaneously exhibits significant differential expression and differential DNA methylation modification. Further research revealed that in LBW placenta... PARP3 The methylation level in the gene promoter region was significantly higher than that in HBW placenta; treatment of the porcine placental trophoblast cell line PTr2 with the DNA methylation inhibitor 5-Aza significantly reduced... PARP3 Promoter methylation levels and upregulation of PARP3 expression confirmed that PARP3 expression is negatively regulated by DNA methylation. Functional experiments showed that overexpression of PARP3 in PTr2 cells promoted cell proliferation and migration and inhibited apoptosis; while in PTr2 cells, it inhibited... PARP3 Expression of this substance can inhibit its proliferation and migration, but promote its apoptosis. Further research revealed that it inhibits apoptosis in PTr2 cells. PARP3 Expression of this substance can significantly inhibit the PI3K-Akt signaling pathway, downregulate the expression of PI3K and mTOR, and thus may affect cell homeostasis and placental developmental potential. In summary, PARP3 By regulating its own expression through DNA methylation and participating in the regulation of placental trophoblast cell biological function via the PI3K-Akt signaling pathway, it influences piglet birth weight. Therefore, targeted regulation... PARP3The methylation status or expression level of [the substance] is expected to become an important strategy for improving placental development in pigs, increasing birth weight and survival rate of piglets, and thus improving the reproductive efficiency of sows.

[0109] This study collected placental samples from four litters of the same sex, specifically high birth weight (HBW) and low birth weight (LBW) Large White piglets. RNA-seq and WGBS sequencing were used to reveal differentially modified DNA methylation genes in the placentas of HBW and LBW litters. PARP3 To investigate the differential regulation and significant differential expression of DNA methylation. PARP3 The effect on the development of placental trophoblast cells (PTr2 cells). Then, by studying the methylation level of this gene, it was found... PARP3 The methylation level of the gene in LBW placenta was significantly higher than that in HBW placenta, suggesting that PARP3 Gene methylation status may be closely related to placental development and fetal growth regulation. Then, it regulates PTr2 cells through DNA methylation. PARP3 The expression of [a specific substance] was investigated, and the results showed that the DNA methylation inhibitor 5-Aza could induce [a specific effect] in PTr2 cells. PARP3 Gene expression. This further confirms that during placental development, PARP3 Gene expression is regulated by DNA methylation, and in PTr2 cells treated with 5-Aza... PARP3 Reduced methylation levels in promoter regions and cells PARP3 Expression was upregulated. This was then achieved by either inhibiting or overexpressing PTr2 cells. PARP3 Gene, results showed overexpression PARP3 Genes can promote PTr2 cell proliferation and migration. PTr2 cells play an important role in placental development; promoting PTr2 cell proliferation and migration can effectively promote placental development and further increase piglet birth weight. Furthermore, inhibiting PTr2 cell proliferation and migration can also contribute to placental development. PARP3 Gene expression significantly inhibits the PI3K-Akt signaling pathway, downregulating PI3K and mTOR gene expression. This indicates regulation... PARP3 Gene expression can also regulate the PI3K-Akt signaling pathway in the placenta to maintain placental homeostasis and development, thereby affecting the birth weight of piglets. Therefore, it is possible to regulate... PARP3 Gene methylation levels or expression levels can regulate the quality of porcine placental development and further improve piglet birth weight; it can also be achieved by regulating... PARP3 Gene methylation levels or expression levels can promote the proliferation and / or migration of porcine placental trophoblast cells; they can also be regulated by... PARP3Gene methylation levels or expression levels regulate the PI3K-Akt signaling pathway in porcine placental trophoblast cells, thereby regulating the proliferation or apoptosis of placental trophoblast cells to maintain placental homeostasis and development, thus affecting the birth weight of piglets. Therefore, by regulating... PARP3 The level of gene methylation or expression can be used to improve the quality of pig placental development, further increase the birth weight and survival rate of piglets, and thus improve the reproductive efficiency of sows and the market competitiveness of breeding farms.

Claims

1. PARP3 Application of genes in regulating porcine placental development; or application in the preparation of products for regulating porcine placental development.

2. PARP3 Non-therapeutic use of the gene in promoting the proliferation and / or migration of porcine placental trophoblast cells; or use in the preparation of products that promote the proliferation and / or migration of porcine placental trophoblast cells.

3. Use according to claim 1 or 2, wherein, The application is achieved by modulating PARP3 the expression level or methylation level of the gene.

4. A non-therapeutic method of improving the quality of porcine placental development, wherein, The method includes adding substances that can promote [the growth of] sow feed or drinking water. PARP3 Agents that can inhibit gene expression may PARP3 Reagents for increasing gene methylation levels; or direct injection into the placenta can promote PARP3 Agents that can inhibit gene expression may PARP3 Reagents for gene methylation levels.

5. A non-therapeutic method of increasing birth weight in swine, wherein, The method is achieved by improving the quality of placental development in pigs, and includes adding substances that promote placental development to the sow's feed or drinking water. PARP3 Agents that can inhibit gene expression may PARP3 Reagents for increasing gene methylation levels; or direct injection into the placenta can promote PARP3 Agents that can inhibit gene expression may PARP3 Reagents for gene methylation levels.

6. A non-therapeutic method of promoting proliferation and / or migration of porcine placental trophoblast cells, wherein, The method comprises injecting or transfecting into pig placental trophoblast cells an agent that promotes PARP3 gene expression or an agent that can inhibit PARP3 methylation levels of the gene.

7. The method according to any one of claims 4-6, wherein, The promotion PARP3 Reagents for gene expression include those that can overexpress gene. PARP3 The gene overexpression plasmid, the nucleotide sequence of which is shown in SEQ ID No:5; can inhibit PARP3 The reagent used to measure gene methylation levels was decitabine.

8. A product which can promote the development of porcine placenta, wherein, The product contains an agent that can promote PARP3 gene expression or an agent that can inhibit PARP3 the level of methylation of a gene.

9. Promote PARP3 Applications of gene expression reagents in the preparation of products that promote porcine placental development; or in the preparation of products that promote the proliferation and / or migration of porcine placental trophoblast cells; or in the preparation of products that increase the birth weight of piglets.

10. Regulation PARP3 Application of gene expression reagents in the preparation of products that regulate the PI3K-Akt pathway in porcine placental trophoblast cells.

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