Method for excavating regulatory element / candidate gene related to porcine muscle development and application

Abstract

The invention discloses a method for excavating regulatory elements related to pig muscle development. The method comprises the following steps: S1, constructing an ATAC library and CUTamp for pig muscle tissues in different periods; carrying out library Tag and sequencing; s2, carrying out sequencing on the sequencing data in the step S1 by utilizing ATAC-seq and CUTamp; carrying out multi-omics data conjoint analysis by virtue of a Tag technology, and mining a muscle development time sequence specific activity regulation element, wherein the ATAC-seq technology is used for identifying chromatin accessibility and the CUTamp; a Tag technology is used for identifying H3K4me1, H3K27ac and H3K27me3 histone protein apparent modification, and two groups of data are subjected to joint analysis to excavate period specific activity regulation elements related to pig muscle development. Through multi-omics conjoint analysis, active regulatory elements in different periods in a porcine embryo skeletal muscle development process can be accurately identified, a gene regulatory network is constructed, data support is provided for research of skeletal muscle development and a regulatory mechanism, a target is provided for pork production character optimization and gene improvement, and the method has a wide application prospect. And the method has a promotion effect on molecular breeding and variety improvement of meat traits of meat pigs, and has a relatively good application value.

Description

Technical Field

[0001] The present invention relates to the fields of genomics and bioinformatics, and in particular to a method and application of mining regulatory elements / candidate genes related to pig muscle development. Background Art

[0002] The development and growth of skeletal muscle determine the yield and quality of meat, and the regulation of the number of muscle fibers during skeletal muscle development plays a decisive role in the meat production characteristics of pigs. The number of muscle fibers in pig skeletal muscle is fixed before the individual is born. After birth, its number remains unchanged while its volume increases day by day. Therefore, analyzing the genetic structure and molecular mechanism of pig skeletal muscle development is of great significance for exploring the key sites of skeletal muscle development.

[0003] Gene expression is subject to complex regulation, mainly including two levels: transcriptional regulation and translational regulation. Transcriptional regulation is mainly achieved through the interaction between transcription factors and promoters, enhancers and transcriptional regulatory factors.

[0004] Although studies have identified active regulatory elements in different cells or tissues, existing technologies still have limitations in analyzing the gene expression regulation mechanism in complex biological processes such as pig muscle development. Existing methods lack the ability to capture the details of dynamic changes in gene expression, and there are challenges in integrating multi-omics data to comprehensively analyze gene regulatory networks. Therefore, it is urgent to study a new method that is convenient for efficient mining or prediction of regulatory elements or candidate genes related to pig muscle development, so as to provide data support and research direction for the improvement of pig muscle traits and breeding. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application for mining regulatory elements / candidate genes related to pig muscle development. Through this method, regulatory elements or candidate genes related to pig muscle development can be mined / predicted, providing data support for the study of skeletal muscle development and regulatory mechanisms, and also providing targets for the optimization of pork production traits and genetic improvement. It also plays an important role in promoting the molecular breeding and variety improvement of meat production traits of meat pigs, and has good application value.

[0006] According to a first aspect of the present invention, a method for mining regulatory elements related to pig muscle development is provided, the method comprising: S1: Sample pig muscle tissue at different stages, construct ATAC library and CUT&Tag library and sequence them; S2: The sequencing data in step S1 are analyzed by multi-omics data joint analysis using ATAC-seq and CUT&Tag technologies to mine the time-specific active regulatory elements of muscle development: the ATAC-seq technology is used to identify chromatin accessibility, and the CUT&Tag technology is used to identify H3K4me1, H3K27ac and H3K27me3 histone epigenetic modifications. The two sets of data are jointly analyzed to mine period-specific active regulatory elements related to pig muscle development. Therefore, this method can efficiently and accurately mine or predict active regulatory elements at different stages of pig embryonic skeletal muscle development, construct a gene regulatory network, provide data support for the study of skeletal muscle development and regulatory mechanisms, and provide targets for the optimization of pork production traits and genetic improvement. It also plays an important role in promoting the molecular breeding and variety improvement of meat pig meat production traits, and has good application value.

[0007] In certain embodiments, the method for using the ATAC-seq technology to identify chromatin accessibility includes: performing quality control filtering on the raw sequencing data, aligning the filtered data to the pig reference genome, deleting repetitive sequences using gatk4, deleting sequences aligned to mitochondria, and using MACS2 to perform peak calling on the samples respectively, evaluating the differences in chromatin accessible regions of pig muscle tissue samples at different periods, and the significant difference criteria are: FDR<0.05 and log2|FC|>1, and using the annotatePeak function in the R package ChIPseeker for peak annotation, and using the clusterProfiler package to perform GO and KEGG analysis on the annotated differential genes, and the significant enrichment criterion is q-value<0.05.

[0008] In certain embodiments, the method for using the CUT&Tag technology to identify H3K4me1, H3K27ac and H3K27me3 histone epigenetic modifications includes: quality control filtering of the raw sequencing data, aligning the filtered data to the pig reference genome, using gatk4 to delete repetitive sequences, deleting sequences aligned to mitochondria, and using MACS2 to perform peak calling on the samples, p<1e-5, evaluating the differences in histone modification levels in pig muscle tissue at different periods, and screening out regions where the histone modification levels in pig muscle tissue at different periods have significantly changed, the significant difference criteria are: FDR<0.05 and log2|FC|>1, using the annotatePeak function in the R package ChIPseeker for peak annotation, and using the clusterProfiler package to perform GO and KEGG analysis on the annotated differential genes, and the significant enrichment criteria are q-value<0.05.

[0009] In certain embodiments, the regulatory element is a promoter and / or an enhancer, and the method for jointly analyzing the two sets of data includes: using Bedtools intersect to combine ATAC-seq data and CUT&Tag data to screen active promoters and enhancers in open chromatin regions: the promoter screening conditions are regions that simultaneously meet the requirements of H3K4me3 enrichment, H3K27ac enrichment, and increased chromatin accessibility; the enhancer screening conditions are regions that simultaneously meet the requirements of H3K4me1 enrichment, H3K27ac enrichment, and increased chromatin accessibility.

[0010] In certain embodiments, the method further comprises using Bedtools subtract to screen out specific active regulatory elements of different periods from the active regulatory elements in the open region of chromatin, and the screening criteria for the specific active regulatory elements are defined as follows: the regulatory elements that are active in a specific period should have strict period specificity, that is, they only appear in this specific period and absolutely do not include active regulatory elements that have appeared in any other period.

[0011] In certain embodiments, the pig muscle tissues at different stages refer to the longissimus dorsi muscle in pig embryos at 60, 70, 90, and 105 days of gestation.

[0012] According to the second aspect of the present invention, a regulatory element related to pig muscle development mined by the above method is provided. Thus, the regulatory element mined by the method is further used to predict candidate genes related to pig muscle traits, and ultimately used to improve pig muscle traits and improve pig meat quality traits.

[0013] According to the third aspect of the present invention, there is provided an application of the above method in mining regulatory elements related to pig muscle development. Thus, through this application, the mined regulatory elements can be further used to predict candidate genes related to pig muscle traits, and ultimately used to improve pig muscle traits and improve pig meat quality traits.

[0014] According to the fourth aspect of the present invention, a method for mining candidate genes related to pig muscle development is provided, the method comprising mining the regulatory elements related to pig muscle development using the above method or using the mined regulatory elements and combining RNA-seq technology to analyze gene expression to mine candidate genes related to pig muscle development, the screening conditions of the candidate genes are: the presence of promoter and / or enhancer regulation; significant changes in gene expression; reports related to muscle development in other species, but no relevant reports of genes in the muscle development process of pigs. Thus, the candidate genes related to pig muscle development that can be mined / predicted by this method can be further used as target genes for improving pig muscle traits, which can improve pig muscle quality, play an important role in promoting molecular breeding and variety improvement of meat pig meat production traits, and have good application value.

[0015] According to the fifth aspect of the present invention, a candidate gene related to pig muscle development mined by the method is provided. Therefore, the candidate gene predicted by the method can be further used as a target gene for improving pig muscle traits, which can improve the muscle quality of pigs, play an important role in promoting molecular breeding and variety improvement of meat production traits of meat pigs, and has good application value.

[0016] According to a sixth aspect of the present invention, the use of KPNA1 and / or UCHL1 genes in regulating the development of pig embryonic skeletal muscle is provided. Thus, the use of the genes KPNA1 and / or UCHL1 in regulating the development of pig embryonic skeletal muscle can improve the muscle quality of pigs, and improve the market competitiveness and application value of pigs.

[0017] Beneficial effects of the present invention: This application can mine / predict regulatory elements / candidate genes related to pig muscle development through a multi-omics joint analysis method. This method can accurately identify active regulatory elements or candidate genes at different stages of pig embryonic skeletal muscle development through a joint analysis of ATAC-seq, CUT&Tag, and RNA-seq technologies, and then construct a gene regulatory network, providing data support for the study of skeletal muscle development and regulatory mechanisms, and also providing targets for the optimization of pork production traits and genetic improvement. It also plays an important role in promoting the molecular breeding and variety improvement of meat pig meat production traits, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Flow chart for multi-omics joint analysis; Figure 2 This is a result diagram of the signal enrichment of ATAC-seq reads of Landrace pig skeletal muscle at different fetal ages on the genome; Figure 3Biological process pathways for genes upregulated by differential chromatin accessibility; Figure 4 The biological process pathways of genes that are differentially upregulated by H3K4me3 modification levels; Figure 5 This is the result diagram of GSEA pathway enrichment analysis; Figure 6 annotate genomic maps for promoters and enhancers; Figure 7 This is the result of period-specific chromatin open region motif analysis; Figure 8 for KPNA1 IGV map of the gene; Fig. 9 for UCHL1 IGV map of the gene. DETAILED DESCRIPTION

[0019] The present application provides a method for mining the timing-specific active regulatory elements of Landrace pig muscle development based on multi-omics joint analysis technology. The method uses ATAC-seq (Assay for Transposase Accessible Chromatinusing sequencing) technology, CUT&Tag (Cleavage Under Targets and Tagmentation) technology and RNA-seq (RNA sequencing) technology for joint analysis, analyzes the longissimus dorsi muscle samples of Landrace pigs at gestational age of 60, 70, 90, and 105 days, identifies the regulatory elements related to pig muscle development, and then further mines / predicts the key candidate genes related to muscle development by mining the regulatory elements. The analysis process is as follows: Figure 1 As shown, the specific steps include: 1. Identify differences in chromatin accessibility based on ATAC-seq technology.

[0020] ATAC libraries were constructed and sequenced for the longissimus dorsi muscle of Landrace pigs at different gestational ages (gestational age 60, 70, 90, and 105 days), and the raw sequencing data were quality-controlled and filtered. The filtered data were aligned to the pig reference genome using Bowtie2 software, and the duplicate sequences were deleted using gatk4, the sequences aligned to mitochondria were deleted, and the samples were peak called using MACS2. The differences in chromatin accessible regions of longissimus dorsi muscle samples of pigs at different gestational ages were evaluated, and the regions with significant changes in chromatin regional accessibility were divided into UP group with increased accessibility and DOWN group with decreased accessibility using the DiffBind package. The significant difference criteria were: FDR<0.05 and log2|FC|>1. The "annotatePeak" function in the R package ChIPseeker was used for peak annotation. The clusterProfiler package was used to perform GO and KEGG analysis on the annotated differentially expressed genes, and the significant enrichment criteria were q-value<0.05.

[0021] The results of identifying differences in chromatin accessibility based on ATAC-seq technology showed that the enrichment of ATAC-seq reads at 70 and 90 days of gestational age at TSS sites was stronger than that at 60 and 105 days of gestational age ( Figure 2 ). Genes in the differential chromatin regions of the promoter regions were selected for GO analysis. The results showed that genes with increased chromatin accessibility in the longissimus dorsi muscle samples of 90-day-old pigs were significantly enriched in biological processes related to muscle development, such as muscle structure development, muscle system processes, muscle cell development, and muscle cell differentiation ( Figure 3 ).

[0022] 2. CUT&Tag technology identifies epigenetic modifications of histones (H3K27ac, H3K4me1, H3K4me3).

[0023] CUT&Tag libraries were constructed and sequenced for the longissimus dorsi muscle of Landrace pigs at different gestational ages (gestational age 60, 70, 90, and 105 days), and the original sequencing data were quality-controlled and filtered. The filtered data were aligned to the pig reference genome using Bowtie2 software, and the duplicate sequences were deleted using gatk4, and the sequences aligned to mitochondria were deleted. MACS2 was used to perform peak calling on the samples, and p<1e-5 was used. The differences in histone modification levels in skeletal muscle of pigs at different gestational ages were evaluated, and the regions with significant changes in histone modification levels in skeletal muscle of pigs at different gestational ages were screened out. The "annotatePeak" function in the R package ChIPseeker was used to divide the regions with significant changes in histone modification levels into the UP group with increased accessibility and the DOWN group with decreased accessibility. The significant difference criteria were: FDR<0.05 and log2|FC|>1. The "annotatePeak" function in the R package ChIPseeker was used for peak annotation. The clusterProfiler package was used to perform GO and KEGG analysis on the annotated differentially expressed genes, and the significant enrichment criterion was q-value < 0.05.

[0024] The results of epigenetic modification of histones (H3K27ac, H3K4me1, H3K4me3) identified based on CUT&Tag technology showed that when CUT&Tag reads were annotated to the genome, H3K4me3 was mainly enriched in the promoter region, H3K27ac was enriched in both the promoter and enhancer regions, and H3K4me1 was mainly enriched in the enhancer region. Through GO analysis, the results showed that genes with increased H3K4me3 modification levels in 90-day-old pig muscles were enriched in cellular components such as myofibrils, contractile fibers, and muscle knots, as well as in biological processes and pathways related to skeletal muscle development such as calcium ions and MAPK signaling pathways ( Figure 4 ).

[0025] 3. Perform joint analysis based on ATAC-seq analysis results and CUT&Tag analysis results.

[0026] Based on the results of ATAC-seq analysis and CUT&Tag analysis, we conducted a joint analysis to mine active regulatory elements in the open chromatin region. The identified regulatory elements are promoters and enhancers. The screening conditions for promoters are regions that simultaneously meet the requirements of H3K4me3 enrichment, H3K27ac enrichment, and increased chromatin accessibility; the screening conditions for enhancers are regions that simultaneously meet the requirements of H3K4me1 enrichment, H3K27ac enrichment, and increased chromatin accessibility. Genes that meet the screening conditions are annotated ( Figure 6), the active promoters identified were mainly located in the proximal promoter region (<=1kb); the active enhancers identified were mainly located in the proximal promoter region (<=1kb), intronic region and distal intergenic region. The regions where active promoters and enhancers were located were extracted, which were the specific active regions. The specific active regions in the longissimus dorsi muscle samples of pigs at different gestational ages were counted at different periods. The results showed that the number of specific active regulatory elements (promoters and enhancers) in the muscles of pigs at gestational ages of 60, 70, 90 and 105 days were 2211, 25226, 17198 and 28276, respectively.

[0027] 4. Analyze gene expression based on RNA-seq technology.

[0028] The RNA extracted from the longissimus dorsi muscle of Landrace pigs at different gestational ages (gestational age 60, 70, 90, and 105 days) was used for library construction and sequencing, and the raw sequencing data was quality controlled and filtered. The filtered data was aligned to the pig reference genome using STAR software, and transcript quantitative analysis was performed using stringtie. The expression matrix was generated using prepDE.py in stringtie; GSEA was used for gene enrichment analysis.

[0029] The results of analyzing the changes in gene expression based on RNA-seq technology showed that compared with 60-day-old pigs, the genes with upregulated expression in the muscles of 90-day-old pigs were significantly enriched in multiple energy metabolism-related pathways, including carbon metabolism, TCA cycle, fatty acid degradation, glycolysis / gluconeogenesis, oxidative phosphorylation, pyruvate metabolism, etc. ( Figure 5 ).

[0030] 5. Identify active regulatory elements in chromatin open regions, period-specific active regions, and specific motif analysis.

[0031] Bedtools intersect was used to combine ATAC-seq data and CUT&Tag data to screen active promoters and enhancers. The screening conditions for promoters were regions that simultaneously met the conditions of H3K4me3 enrichment, H3K27ac enrichment, and increased chromatin accessibility. The screening conditions for enhancers were regions that simultaneously met the conditions of H3K4me1 enrichment, H3K27ac enrichment, and increased chromatin accessibility. Bedtools subtract was used to screen out specific active regulatory elements of different periods from the active regulatory elements in the chromatin open region (the data of each developmental stage except the specific developmental stage were extracted and their union was constructed; then, the difference between the data of a specific period and the union of the data of other periods was calculated, and the result obtained was the specific active regulatory region of the specific period. On this basis, through further screening processes, the specific active regulatory elements corresponding to different periods were identified from the specific active regulatory region), and the information of period-specific active regions was counted. Peaks of period-specific active regions were screened, and motif analysis was performed using Homer. In summary, the screening criteria for specific active regulatory elements are defined as follows: regulatory elements that are active during a specific period should have strict period specificity, that is, they only appear in that specific period and absolutely do not include active regulatory elements that have appeared in any other period.

[0032] The "findMotifsGenome.pl" program in the HOMER software package was used to perform motif analysis on the obtained period-specific active regions. The chromatin accessible regions in the longissimus dorsi muscle samples of gestational age 90 and 105 days were significantly enriched with motif sequences of transcription factors such as Mef2c, CTCF, and NFIX, as well as muscle development-related regulatory factors ( Figure 7 ).

[0033] 6. Combined analysis to identify specific gene expression.

[0034] The annotated gene information of specific active regions and non-specific active regions in different periods were counted separately. Combined with RNA-seq data, gene differential expression analysis was performed based on different periods to obtain period-specific gene expression changes, so as to explore / predict key candidate genes related to pig muscle development.

[0035] The ATAC-seq results showed that the chromatin accessibility of the promoter regions of a large number of genes related to muscle development regulation increased at 90 days of gestation. The CUT&Tag results showed that the genes with increased H3K4me3 modification levels at 90 days of gestation were enriched in the biological processes related to skeletal muscle development. The RNA-seq results showed that the expression levels of a large number of energy metabolism-related genes were upregulated at 90 days of gestation, indicating that 90 days of gestation may be a critical time point for embryonic development. Combining the period-specific active regions and the gene expression change results obtained by RNA-seq, the gene expression of the specific active regions was analyzed. Based on the gene expression at 90 days of gestation, the genes were compared and analyzed with those at 60 and 70 days of gestation, and the genes that met the following requirements were screened out: the presence of promoter and / or enhancer regulation; significant changes in gene expression; reports related to muscle development have been reported in other species, but there are no related reports in the process of pig embryonic muscle development. The results showed that the gene that was regulated by both promoters and enhancers and met the above requirements was KPNA1 ( Figure 8 ) and UCHL1 ( Fig. 9 ), these two genes may be potential key candidate genes for pig embryonic skeletal muscle development. Among them, the literature Karyopherin Alpha 1 Regulates Satellite Cell Proliferation and Survival by Modulating Nuclear Import reported that the KPNA1 gene is related to mouse muscle development, and the literature UCHL1 Regulates Lipid and Perilipin 2 Level in Skeletal Muscle reported that the UCHL1 gene is related to mouse muscle development.

[0036] This method can be used to efficiently and accurately mine / predict regulatory elements / candidate genes related to pig muscle development. The mined regulatory elements / candidate genes can be used to construct gene regulatory networks, provide data support for the study of skeletal muscle development and regulatory mechanisms, and provide targets for the optimization of pork production traits and genetic improvement. It also plays an important role in promoting molecular breeding and variety improvement of meat production traits of meat pigs and has good application value.

Claims

1. A method for mining regulatory elements related to pig muscle development, wherein: The method comprises: S1: Sample pig muscle tissue at different stages, construct ATAC library and CUT&Tag library and sequence them; S2: Use ATAC-seq and CUT&Tag technologies to perform multi-omics data joint analysis on the sequencing data in step S1 to explore the timing-specific active regulatory elements of muscle development: the ATAC-seq technology is used to identify chromatin accessibility, and the CUT&Tag technology is used to identify H3K4me1, H3K27ac and H3K27me3 histone epigenetic modifications. The two sets of data are jointly analyzed to explore the period-specific active regulatory elements related to pig muscle development.

2. The method according to claim 1, wherein: The method for using the ATAC-seq technology to identify chromatin accessibility includes: quality control filtering of raw sequencing data, aligning the filtered data to a pig reference genome, deleting duplicate sequences using gatk4, deleting sequences aligned to mitochondria, and performing peak calling on samples using MACS2 to evaluate differences in chromatin accessible regions of pig muscle tissue samples at different stages, with significant differences being: FDR < 0.05 and log2|FC| > 1, and using the annotatePeak function in the R package ChIPseeker for peak annotation, and using the clusterProfiler package to perform GO and KEGG analysis on the annotated differentially expressed genes, with a significant enrichment criterion of q-value < 0.

05.

3. The method according to claim 2, wherein: The method for using the CUT&Tag technology to identify H3K4me1, H3K27ac and H3K27me3 histone epigenetic modifications includes: performing quality control filtering on the original sequencing data, aligning the filtered data to the pig reference genome, deleting repeated sequences using gatk4, deleting sequences aligned to mitochondria, and using MACS2 to perform peak calling on the samples, p<1e-5, evaluating the differences in histone modification levels in pig muscle tissue at different periods, screening out regions where the histone modification levels in pig muscle tissue at different periods have significantly changed, and the significant difference criteria are: FDR <0.05 and log2|FC| > 1, using the annotatePeak function in the R package ChIPseeker to perform peak annotation, and using the clusterProfiler package to perform GO and KEGG analysis on the annotated differential genes, and the significant enrichment criterion is q-value <0.

05.

4. The method according to claim 3, wherein: The regulatory elements are promoters and / or enhancers, and the method for joint analysis of the two sets of data includes: using Bedtools intersect to combine ATAC-seq data and CUT&Tag data to screen active promoters and enhancers in chromatin open areas: the promoter screening conditions are regions that simultaneously meet H3K4me3 enrichment, H3K27ac enrichment, and increased chromatin accessibility; the enhancer screening conditions are regions that simultaneously meet H3K4me1 enrichment, H3K27ac enrichment, and increased chromatin accessibility.

5. The method according to claim 4, wherein: The method also includes using Bedtools subtract to screen out specific active regulatory elements of different periods from the active regulatory elements in the open region of chromatin. The screening criteria for the specific active regulatory elements are defined as follows: the regulatory elements that are active in a specific period should have strict period specificity, that is, they only appear in this specific period and absolutely do not include active regulatory elements that have appeared in any other period.

6. Regulatory elements related to pig muscle development mined using the method described in any one of claims 1-5.

7. Application of the method described in any one of claims 1 to 5 in exploring regulatory elements associated with pig muscle development.

8. A method for mining candidate genes related to pig muscle development, wherein: The method comprises mining candidate genes related to pig muscle development by using the method described in any one of claims 1 to 5 or mining candidate genes related to pig muscle development by using the regulatory elements described in claim 6 in combination with RNA-seq technology to analyze gene expression. The screening conditions of the candidate genes are: the presence of promoter and / or enhancer regulation; significant changes in gene expression; and genes that have been reported to be related to muscle development in other species but have not been reported to be related to pig muscle development.

9. Candidate genes related to pig muscle development mined using the method described in claim 8.

10. Application of KPNA1 and / or UCHL1 genes in regulating pig embryonic skeletal muscle development.

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