CN116343929B - Method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology and application - Google Patents
Method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology and application Download PDFInfo
- Publication number
- CN116343929B CN116343929B CN202310613009.9A CN202310613009A CN116343929B CN 116343929 B CN116343929 B CN 116343929B CN 202310613009 A CN202310613009 A CN 202310613009A CN 116343929 B CN116343929 B CN 116343929B
- Authority
- CN
- China
- Prior art keywords
- peak
- sheep
- skeletal muscle
- difference
- data
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 126
- 241001494479 Pecora Species 0.000 title claims abstract description 92
- 210000002027 skeletal muscle Anatomy 0.000 title claims abstract description 52
- 238000011161 development Methods 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000005516 engineering process Methods 0.000 title claims abstract description 17
- 238000012163 sequencing technique Methods 0.000 claims abstract description 24
- 210000003205 muscle Anatomy 0.000 claims abstract description 20
- 238000001114 immunoprecipitation Methods 0.000 claims abstract description 15
- 238000003908 quality control method Methods 0.000 claims abstract description 13
- 210000001142 back Anatomy 0.000 claims abstract description 10
- 238000010276 construction Methods 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 8
- 230000018109 developmental process Effects 0.000 claims description 41
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 26
- 238000004458 analytical method Methods 0.000 claims description 20
- 230000014509 gene expression Effects 0.000 claims description 18
- 238000010201 enrichment analysis Methods 0.000 claims description 17
- 230000001105 regulatory effect Effects 0.000 claims description 16
- 230000019491 signal transduction Effects 0.000 claims description 16
- 108020004999 messenger RNA Proteins 0.000 claims description 15
- 238000005065 mining Methods 0.000 claims description 12
- 230000011987 methylation Effects 0.000 claims description 11
- 238000007069 methylation reaction Methods 0.000 claims description 11
- 230000004060 metabolic process Effects 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 238000005096 rolling process Methods 0.000 claims description 5
- 108010066587 tRNA Methyltransferases Proteins 0.000 claims description 5
- 102000018477 tRNA Methyltransferases Human genes 0.000 claims description 5
- 230000007730 Akt signaling Effects 0.000 claims description 4
- ZEOWTGPWHLSLOG-UHFFFAOYSA-N Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F Chemical compound Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F ZEOWTGPWHLSLOG-UHFFFAOYSA-N 0.000 claims description 4
- 101100193652 Dictyostelium discoideum rapA gene Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102100035307 Fibroblast growth factor 16 Human genes 0.000 claims description 4
- 108050002072 Fibroblast growth factor 16 Proteins 0.000 claims description 4
- 102100023593 Fibroblast growth factor receptor 1 Human genes 0.000 claims description 4
- 101710182386 Fibroblast growth factor receptor 1 Proteins 0.000 claims description 4
- 101000916644 Homo sapiens Macrophage colony-stimulating factor 1 receptor Proteins 0.000 claims description 4
- 102100028198 Macrophage colony-stimulating factor 1 receptor Human genes 0.000 claims description 4
- 238000003559 RNA-seq method Methods 0.000 claims description 4
- 102000056172 Transforming growth factor beta-3 Human genes 0.000 claims description 4
- 108090000097 Transforming growth factor beta-3 Proteins 0.000 claims description 4
- 108020004417 Untranslated RNA Proteins 0.000 claims description 4
- 102000039634 Untranslated RNA Human genes 0.000 claims description 4
- 239000011324 bead Substances 0.000 claims description 4
- 238000009826 distribution Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 101150046266 foxo gene Proteins 0.000 claims description 4
- 238000002372 labelling Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 101150056532 terf2ip gene Proteins 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 102000010029 Homer Scaffolding Proteins Human genes 0.000 claims description 3
- 108010077223 Homer Scaffolding Proteins Proteins 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 230000002060 circadian Effects 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 3
- 229940088597 hormone Drugs 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 230000004096 skeletal muscle tissue growth Effects 0.000 claims description 3
- 230000012488 skeletal system development Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 235000013372 meat Nutrition 0.000 abstract description 5
- 230000007246 mechanism Effects 0.000 abstract description 5
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 230000009025 developmental regulation Effects 0.000 abstract description 4
- 230000006872 improvement Effects 0.000 abstract description 4
- 230000026267 regulation of growth Effects 0.000 abstract description 4
- 238000003556 assay Methods 0.000 abstract description 3
- 230000001973 epigenetic effect Effects 0.000 abstract description 2
- 238000012986 modification Methods 0.000 description 12
- 230000004048 modification Effects 0.000 description 11
- 230000035935 pregnancy Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 235000019687 Lamb Nutrition 0.000 description 4
- 230000003828 downregulation Effects 0.000 description 4
- 241000283903 Ovis aries Species 0.000 description 3
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 3
- 108091036078 conserved sequence Proteins 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101000967135 Homo sapiens N6-adenosine-methyltransferase catalytic subunit Proteins 0.000 description 2
- 101001013582 Homo sapiens N6-adenosine-methyltransferase non-catalytic subunit Proteins 0.000 description 2
- 101000914035 Homo sapiens Pre-mRNA-splicing regulator WTAP Proteins 0.000 description 2
- 108700011325 Modifier Genes Proteins 0.000 description 2
- 102100040619 N6-adenosine-methyltransferase catalytic subunit Human genes 0.000 description 2
- 102100031578 N6-adenosine-methyltransferase non-catalytic subunit Human genes 0.000 description 2
- 102100026431 Pre-mRNA-splicing regulator WTAP Human genes 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 101100214180 Drosophila melanogaster Ythdf gene Proteins 0.000 description 1
- 101100278476 Homo sapiens DPP10 gene Proteins 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 101000666873 Homo sapiens Protein virilizer homolog Proteins 0.000 description 1
- 101000959153 Homo sapiens RNA demethylase ALKBH5 Proteins 0.000 description 1
- 101000744745 Homo sapiens YTH domain-containing family protein 2 Proteins 0.000 description 1
- 101000744718 Homo sapiens YTH domain-containing family protein 3 Proteins 0.000 description 1
- 101000976373 Homo sapiens YTH domain-containing protein 1 Proteins 0.000 description 1
- 101001012529 Homo sapiens rRNA N6-adenosine-methyltransferase METTL5 Proteins 0.000 description 1
- 102100040449 Inactive dipeptidyl peptidase 10 Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 102100038288 Protein virilizer homolog Human genes 0.000 description 1
- 102100039083 RNA demethylase ALKBH5 Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 230000006093 RNA methylation Effects 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102100039644 YTH domain-containing family protein 2 Human genes 0.000 description 1
- 102100039674 YTH domain-containing family protein 3 Human genes 0.000 description 1
- 102100023905 YTH domain-containing protein 1 Human genes 0.000 description 1
- 230000011759 adipose tissue development Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000027288 circadian rhythm Effects 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102100029739 rRNA N6-adenosine-methyltransferase METTL5 Human genes 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/30—Detection of binding sites or motifs
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
- G16B35/10—Design of libraries
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
Abstract
The invention provides a method for excavating sheep skeletal muscle development related genes by MeRIP-Seq technology and application thereof, firstly sequentially carrying out RNA specificity m on extracted sheep dorsum longus muscle tissue RNA 6 A antibody immunoprecipitation, library construction and sequencing, performing quality control filtration on the obtained original data, comparing the filtered data with sheep reference genome data, and performing m on the genome comparison position data 6 Identification and annotation of A peak to obtain the difference m 6 A peak-related gene, and for the difference m 6 The A peak related gene was identified and GO and KEGG functional enrichment assays were performed. The invention provides scientific basis for exploring the growth and development regulation mechanism of sheep skeletal muscle from the aspect of epigenetic, and has important reference significance for molecular breeding and variety improvement of sheep meat production traits.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology and application thereof.
Background
Mutton is favored by consumers due to high protein content and low cholesterol, especially in recent years, the yield of mutton is increased year by year, the consumption proportion of resident mutton is increased, however, the low mutton yield of mutton sheep is one of important factors restricting the production of mutton sheep. The growth and development of skeletal muscle of sheep determine the formation of high-yield and high-quality characters of mutton sheep, and the deep analysis of the regulation and control mechanism of the growth and development of muscle is of great significance for improving the meat production performance of domestic animals.
m 6 The A modification (N6-methylidenosine), i.e.methylation at N sixth of the ribonucleotide adenylate, is the most common post-transcriptional modification of eukaryotic mRNA and accounts for 80% of the RNA methylation modifications. The recognition site for most RNA methylases is predominantly the conserved sequence 5'-RRACH-3' (r=a or G, h= A, T or C). m is m 6 The A methylation modification has been shown to be reversible, and is co-involved with methyltransferases (Writers), demethylases (Erasers), and methylation reading proteins (Readers), etc. Writers mainly comprises METTL3, METTL5, METTL14, KIAA1492, WTAP, etc., and is capable of mediating methylation modification of N at position six of RNA adenylate, wherein METTL3 and METTL14 form heterocomplexes that modify adenylate with WTAP (also called Fl (2) d) and other factors such as KIAA1429 (also called Virilizer). Erasers mainly comprises FTO, ALKBH5 and other homologous genes, and mediates m 6 A demethylation modification. Readers essentially comprise the YTH family (YTHDF 1, YTHDF2, YTHDF3, YTHDC1 and YTHDC 2), eIF3, hnRNA and other homologous genes, by recognizing m 6 A is modified and participates in the biological processes of translation, degradation and the like of downstream RNA. Several studies have shown that m 6 The A can accelerate the processing time of the mRNA precursor, accelerate the transfer speed and the nuclear emergence speed of mRNA in cells, and plays an important role in biological processes such as embryo development, muscle development, adipogenesis, diseases and the like.
Disclosure of Invention
The invention aims to provide a method for excavating skeletal muscle development related genes of sheep by MeRIP-Seq technology and application thereof, and m related to skeletal muscle development is identified by the back longest muscle MeRIP-Seq of key development stage of sheep 6 The A modified gene provides scientific basis for exploring the growth and development regulation mechanism of sheep skeletal muscle, and has important reference significance for molecular breeding and variety improvement of sheep meat production traits.
The invention provides a MeRIP-Seq-based technologyA method of mining a skeletal muscle development related gene of a sheep comprising: sequentially performing RNA specificity m on the extracted sheep dorsum longus muscle tissue RNA 6 A, immunoprecipitation of antibodies, library construction and sequencing; performing quality control filtering on the raw data obtained by sequencing to obtain filtered data, and comparing the filtered data with sheep reference genome data to obtain genome comparison position data; m-comparing the genomic alignment position data 6 Authentication and annotation of A peak to obtain m 6 An A peak-related gene; according to said m 6 A peak-related genes give a difference m 6 A peak-related gene, and for the difference m 6 The A peak related gene was identified and GO and KEGG functional enrichment assays were performed.
According to the method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology provided by the invention, the extracted sheep dorsum longus muscle tissue RNA is sequentially subjected to RNA specificity m 6 A antibody immunoprecipitation, library construction, and sequencing, including: enrichment of extracted sheep dorsum longus muscle RNA by VAHTS mRNA capture beads and ZnCl addition 2 Incubation at 95℃for 5min-10min for RNA-specific m 6 A, immunoprecipitation of the antibody; a chain RNA sequencing library was constructed using the Illunmia-locked KC-DigitalTM chain mRNA library preparation kit.
According to the method for mining sheep skeletal muscle development related genes based on the MeRIP-Seq technology, the quality control filtering is carried out on the raw data obtained by sequencing to obtain filtered data, and the filtered data is compared with sheep reference genome data to obtain genome comparison position data, and the method comprises the following steps: carrying out quality statistics on the raw data obtained by sequencing through FastQC software; performing quality control by using NGSQC Toolkit to obtain the filtered data; and comparing the filtered data with the sheep reference genome data by using bowtie2 comparison software to obtain the genome comparison position data.
According to the method for mining sheep skeletal muscle development related genes based on MeRIP-Seq technology, the genome is subjected to m comparison position data 6 Authentication and annotation of A peak to obtain m 6 An apeak related gene comprising: peak rolling scan using exomePeak, analyze m 6 The position of the A peak and the corresponding gene, and the threshold value is q value<0.05; using bedtools vs. m 6 Labeling the peak A; the m is treated with deep tools 6 Analyzing the peak value distribution of the peak A; the m is 6 The difference in peak a was determined by python script using a fisher test; merging adjacent significant enrichment regions into one enrichment region as the final m 6 Apeak; extraction of m Using Homer 6 The sequence of the interval in which A peak is located, for m 6 Scanning the common motif among the A peaks, and searching for a common motif region; m between repeated samples in a group 6 A peak filters, retains overlay>50% of total m 6 A peak related gene.
The method for mining sheep skeletal muscle development related genes based on MeRIP-Seq technology provided by the invention is characterized in that the gene is expressed by the m 6 A peak-related genes give a difference m 6 A peak-related gene, and for the difference m 6 The A peak related gene is identified and GO and KEGG functional enrichment analysis is carried out, which comprises the following steps: according to said m 6 A peak related gene, fold Change>2，P<0.05 is the screening difference m 6 A peak related gene standard, obtaining the difference m 6 An A peak-related gene; the difference m is treated with KOBASA 6 The A peak related gene was subjected to GO and KEGG functional enrichment analysis.
The invention also provides m 6 Use of the apeak gene in sheep skeletal muscle development for non-disease diagnostic purposes.
M provided according to the invention 6 Application of A peak gene in development of skeletal muscle of sheep and skeletal muscle m of sheep in different development periods 6 The motif of A corresponds to the motif RRACH.
M provided according to the invention 6 Application of A peak gene in development of skeletal muscle of sheep, and difference m of skeletal muscle of sheep in different development periods 6 The GO analysis of A expressed peak annotated genes are enriched for protein binding, enzyme binding and developmental processes; cotton for different development periodSheep skeletal muscle difference m 6 KEGG analysis of a gene annotated to express upregulated peak is enriched in circadian, hormone-regulated signaling pathways; skeletal muscle difference m of sheep at different developmental stages 6 GO analysis of A expressed down-regulated peak annotated genes were enriched for tRNA metabolic processes, RNA methylase activity and ncRNA metabolic processes; skeletal muscle difference m of sheep at different developmental stages 6 KEGG analysis of a genes annotated for peak down-regulated expression were enriched in PI3K-Akt signaling pathway, rap1 signaling pathway, foxO signaling pathway and cGMP-PKG signaling pathway.
M provided according to the invention 6 Application of A peak gene in sheep skeletal muscle development, low m involved in sheep skeletal muscle growth and development 6 The A methylation high expression genes are TGFB3, FGF16, FGFR1 and CSF1R.
The invention provides a method for excavating sheep skeletal muscle development related genes by MeRIP-Seq technology and application thereof, wherein the method comprises the following steps: sequentially performing RNA specificity m on the extracted sheep dorsum longus muscle tissue RNA 6 A, immunoprecipitation of antibodies, library construction and sequencing; performing quality control filtering on the raw data obtained by sequencing to obtain filtered data, and comparing the filtered data with sheep reference genome data to obtain genome comparison position data; performing m on genome alignment position data 6 Authentication and annotation of A peak to obtain m 6 An A peak-related gene; according to m 6 A peak-related genes give a difference m 6 A peak-related gene, and for the difference m 6 Identification of A peak related Gene and GO and KEGG functional enrichment analysis the invention identifies m related to skeletal muscle development from an epigenetic perspective by the dorsi-longest muscle MeRIP-Seq at key developmental stage of sheep 6 The A modified gene provides scientific basis for exploring the growth and development regulation mechanism of sheep skeletal muscle, and has important reference significance for molecular breeding and variety improvement of sheep meat production traits.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart of a method for mining sheep skeletal muscle development related genes based on MeRIP-Seq technology;
FIG. 2 is a schematic diagram of m of D40 and F70 provided by the present invention 6 A peak number graph;
FIG. 3 is a diagram of m provided by the present invention 6 A peak profile;
FIG. 4 is a graph of the number of genes D40 and F70 provided by the present invention;
FIG. 5 is a diagram of a motif conserved sequence provided by the present invention;
FIG. 6 is a graph of GO enrichment analysis of differential up-regulated expression peak annotation genes provided by the present invention;
FIG. 7 is a KEGG enrichment analysis of the differential up-regulated expression peak annotation gene provided by the present invention;
FIG. 8 is a graph of GO enrichment analysis of differential downregulation expression peak annotation genes provided by the present invention;
FIG. 9 is a KEGG enrichment analysis of differential downregulation expression peak annotation genes provided by the present invention;
FIG. 10 is a graph of m for D40 and F70 provided by the present invention 6 Wien diagram of A modified gene;
FIG. 11 is a graph of m for D40 and F70 provided by the present invention 6 Four-quadrant plots of panel a and mRNA panel.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following describes a method for mining sheep skeletal muscle development related genes by MeRIP-Seq technology and application thereof with reference to FIGS. 1-11.
Referring to fig. 1, fig. 1 is a schematic flow chart of a method for mining skeletal muscle development related genes of sheep based on the MeRIP-Seq technique provided by the invention.
The invention provides a method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology, which comprises the following steps:
101: sequentially performing RNA specificity m on the extracted sheep dorsum longus muscle tissue RNA 6 A, immunoprecipitation of antibodies, library construction and sequencing;
102: performing quality control filtering on the raw data obtained by sequencing to obtain filtered data, and comparing the filtered data with sheep reference genome data to obtain genome comparison position data;
103: performing m on genome alignment position data 6 Authentication and annotation of A peak to obtain m 6 An A peak-related gene;
104: according to m 6 A peak-related genes give a difference m 6 A peak-related gene, and for the difference m 6 The A peak related gene was identified and GO and KEGG functional enrichment assays were performed.
The invention selects three gestational sheep of 70 days old and 40 days old lambs after birth, collects the longus muscle tissue of the back to carry out transcriptome MeRIP-Seq sequencing, and integrates and analyzes m 6 A transcriptome and mRNA transcriptome data, searching genes and regulatory pathways related to skeletal muscle of sheep, and identifying m related to skeletal muscle development by the invention through the dorsi-longus muscle MeRIP-Seq of key development stage of sheep 6 The A modified gene provides scientific basis for exploring the growth and development regulation mechanism of sheep skeletal muscle, and has important reference significance for molecular breeding and variety improvement of sheep meat production traits.
Based on the above embodiments:
as a preferred embodiment, RNA-specific m is sequentially performed on the extracted sheep longus muscle tissue RNA 6 A antibody immunoprecipitation, library construction, and sequencing, including: enrichment of extracted sheep dorsum longus muscle tissue RNA by VAHTS mRNA capture beadsCollecting and adding ZnCl 2 Incubation at 95℃for 5min-10min for RNA-specific m 6 A, immunoprecipitation of the antibody; a chain RNA sequencing library was constructed using the Illunmia-locked KC-DigitalTM chain mRNA library preparation kit.
(1) According to the invention, a 70-day-old multiple-wave foetus sheep is used as a control group and 40-day-old multiple-wave lambs after birth are used as experimental groups, healthy 3 female sheep are randomly selected for slaughtering, the longus dorsi muscle of the foetus sheep and the longus dorsi muscle of the lambs are collected, and muscle tissues are stored in liquid nitrogen for RNA extraction.
(2) Enrichment with 50ug total RNA was performed by VAHTS mRNA capture beads (VAHTS, catalog No. N401-01/02); addition of 20mM ZnCl to mRNA 2 Incubating at 95℃for 5-10 min until the RNA is broken into short fragments of about 100-200 nt; 10% of the RNA fragments were then saved as "Input" and the remaining fragments were used for m 6 A Immunoprecipitation (IP); by means of specificity m 6 Antibodies a (synthetic Systems, 202203) were subjected to co-immunoprecipitation; RNA samples of Input and IP were prepared using TRIzol reagent (Invitrogen).
(3) A chain RNA sequencing library was constructed using the Illunmia-mounted KC-DigitalTM chain mRNA library preparation kit (catalog number DR08502, wohan Kangji test, china). The kit eliminates repetitive bias in PCR and sequencing steps by labeling pre-amplified cDNA molecules with a unique analytical identifier (UMI) of 8 random bases; enriching library products corresponding to 200-500bps, quantifying the library by using Qubit3.0, and determining whether the library concentration is suitable for the machine; the street-linker was purified with magnetic beads and the products were PCR amplified.
As a preferred embodiment, performing quality control filtering on raw data obtained by sequencing to obtain filtered data, and comparing the filtered data with reference genome data of sheep to obtain genome comparison position data, including: carrying out quality statistics on the raw data obtained by sequencing through FastQC software; performing quality control by using NGSQC Toolkit to obtain filtered data; and comparing the filtered data with sheep reference genome data by using bowtie2 comparison software to obtain genome comparison position data.
Specifically, the raw data obtained after IIIomina sequencing is converted into sequence data, namely FASTQ format, through Base rolling. The quality control by using FASTQC software mainly comprises removing joints, low-quality data, UID deduplication analysis, ribosome reads and the like, and finally obtaining high-quality data Clean data information, namely filtered data.
The data were first aligned to a sheep ribosome database using Bowtie2 software, and reads of the aligned ribosomes were removed. Then comparing the reads obtained in the last step with sheep reference genome (oar_rambouillet_v1.0) for analysis to obtain data with unique comparison positions; the resulting clean reads were visually browsed using IGV software.
The NGSQC Toolkit version may be, but is not limited to, v2.3.3.
As a preferred embodiment, the genomic alignment position data is subjected to m 6 Authentication and annotation of A peak to obtain m 6 An apeak related gene comprising: peak rolling scan using exomePeak, analyze m 6 The position of the A peak and the corresponding gene, and the threshold value is q value<0.05; using bedtools vs. m 6 Labeling the peak A; using deep tools vs. m 6 Analyzing the peak value distribution of the peak A; m is m 6 The difference in peak a was determined by python script using a fisher test; merging adjacent significant enrichment regions into one enrichment region as the final m 6 Apeak; extraction of m Using Homer 6 Sequence pair m of interval where A peak is located 6 Scanning the common motif among the A peaks, and searching for a common motif region; m between repeated samples in a group 6 A peak filters, retains overlay>50% of total m 6 A peak related gene.
exomePeak version may be, but is not limited to v3.8; the bedtools version may be, but is not limited to, v2.24.0; the deep tools version may be, but is not limited to, v2.4.1; the Home version may be, but is not limited to v4.10.
As a preferred embodiment, according to m 6 A peak-related genes give a difference m 6 A peak-related gene, and for the difference m 6 Phase A peakThe related genes are identified, and GO and KEGG functional enrichment analysis is carried out, which comprises the following steps: according to m 6 A peak related gene, fold Change>2，P<0.05 is the screening difference m 6 A peak related gene standard, obtaining the difference m 6 An A peak-related gene; use of KOBAS for variance m 6 The A peak related gene was subjected to GO and KEGG functional enrichment analysis.
The KOBAS version may be, but is not limited to, v2.2.1.
The invention also provides m 6 The application of the A peak gene in sheep skeletal muscle development is non-disease diagnosis.
Double-ended sequencing was performed on 12 libraries using the MeRIP-Seq sequencing method, and data quality control and genome alignment results are shown in table 1:
TABLE 1 MeRIP-Seq sequencing and alignment data overview
Note that: d40 represents 40-day-old lamb, and F70 represents gestational 70-day-old foetus sheep
Referring to FIG. 2, FIG. 2 shows m of D40 and F70 according to the present invention 6 A peak number graph.
Wherein F70 is gestational sheep of 70 days of gestation, and D40 is postnatal lamb of 40 days of gestation.
Referring to FIG. 3, FIG. 3 shows m of the present invention 6 Apeak profile.
Referring to FIG. 4, FIG. 4 is a graph showing the number of genes D40 and F70 provided by the present invention.
Wherein F70 is gestational sheep of 70 days of gestation, and D40 is postnatal lamb of 40 days of gestation.
Referring to fig. 5, fig. 5 is a schematic conserved sequence chart of the motif provided by the present invention.
As a preferred embodiment, sheep skeletal muscle m is selected from different developmental stages 6 The motif of A corresponds to the motif RRACH.
Peak rolling scans were performed using exomepeaak (v 3.8) software, and a total of 28206 m were found 6 A peak regions, m 6 A peak annotationUp to 11569 genes, D40 having 22027 m 6 Apeak region, F70 has 13153 m 6 Apeak region (fig. 2). These m 6 Apeak is mainly focused on the CDS and 3' utr regions near the stop codon (fig. 3). For m 6 A peak notes and looks for related genes, annotated to 17729 genes altogether, with D40 annotated to 10608 genes and F70 annotated to 7121 genes (FIG. 4). Furthermore, the motif found to be consistent with the classical motif RRACH at different stages in sheep (fig. 5).
Referring to fig. 6, fig. 6 is a graph of GO enrichment analysis of differential up-regulated expression peak annotation genes provided by the present invention.
Referring to FIG. 7, FIG. 7 is a KEGG enrichment analysis chart of the differential up-regulated expression peak annotation gene provided by the present invention.
Referring to fig. 8, fig. 8 is a graph of GO enrichment analysis of differential downregulation expression peak annotation genes provided by the present invention.
Referring to FIG. 9, FIG. 9 is a KEGG enrichment analysis chart of the differential downregulation expression peak annotation gene provided by the present invention.
As a preferred embodiment, sheep skeletal muscle differences m between different developmental stages 6 The GO analysis of A expressed peak annotated genes are enriched for protein binding, enzyme binding and developmental processes; skeletal muscle difference m of sheep at different developmental stages 6 KEGG analysis of a gene annotated to express upregulated peak is enriched in circadian, hormone-regulated signaling pathways; skeletal muscle difference m of sheep at different developmental stages 6 GO analysis of A expressed down-regulated peak annotated genes were enriched for tRNA metabolic processes, RNA methylase activity and ncRNA metabolic processes; skeletal muscle difference m of sheep at different developmental stages 6 KEGG analysis of a genes annotated for peak down-regulated expression were enriched in PI3K-Akt signaling pathway, rap1 signaling pathway, foxO signaling pathway and cGMP-PKG signaling pathway.
To elucidate RNA m 6 Potential function of A methylation in sheep skeletal muscle development, for m 6 Genes with significantly altered levels of a methylation were subjected to gene function enrichment analysis. GO analysis showed peak injection with up-regulated differential expression at different developmental stagesThe released genes are enriched in protein binding, enzyme binding, developmental processes, etc. (FIG. 6). KEGG analysis showed that genes annotated for peak differentially expressed up-regulation at different developmental stages were enriched in signaling pathways of circadian rhythm, hormonal regulation, etc. (fig. 7). For genes annotated by differential expression down-regulated peak, GO analysis results were mainly enriched in tRNA metabolic processes, RNA methylase activity, ncRNA metabolic processes, etc. (fig. 8), KEGG analysis results were mainly enriched in PI3K-Akt signaling pathway, rap1 signaling pathway, foxO signaling pathway, cGMP-PKG signaling pathway, etc. (fig. 9).
Referring to FIG. 10, FIG. 10 shows m of D40 and F70 according to the present invention 6 Wien diagram of the a modification gene.
Referring to FIG. 11, FIG. 11 shows m of D40 and F70 according to the present invention 6 Four-quadrant plots of panel a and mRNA panel.
Wherein F70 is gestational sheep of 70 days of gestation, and D40 is postnatal lamb of 40 days of gestation.
As a preferred embodiment, the low m is involved in the growth and development of skeletal muscle of sheep 6 The A methylation high expression genes are TGFB3, FGF16, FGFR1 and CSF1R.
To analyze m in skeletal muscle development process 6 Dynamic variation of A modification for different periods m 6 A modification gene was identified and found to have 6156 m in common in both phases 6 A modifier gene, D40 specificity m 6 4450A modified genes and F70 specificity m 6 963 a-modified genes (fig. 10). Then, these m-containing compounds are identified 6 In the case of distribution of A-modification related genes in transcriptomes, the Input set data was compared with the IP set (m 6 A modification group) data were subjected to a joint analysis to obtain m 6 Relationship of A methylation modifier gene to gene expression trend (FIG. 11). Identification of low m 6 The A methylation high expression genes include genes such as TGFB3, FGF16, FGFR1, CSF1R and the like which are involved in skeletal muscle growth and development.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. A method for mining sheep skeletal muscle development related genes based on the MeRIP-Seq technology, comprising:
sequentially performing RNA specificity m on the extracted sheep dorsum longus muscle tissue RNA 6 A, immunoprecipitation of antibodies, library construction and sequencing;
performing quality control filtering on the raw data obtained by sequencing to obtain filtered data, and comparing the filtered data with sheep reference genome data to obtain genome comparison position data;
m-comparing the genomic alignment position data 6 Authentication and annotation of A peak to obtain m 6 An A peak-related gene;
according to said m 6 A peak-related genes give a difference m 6 A peak-related gene, and for the difference m 6 Identifying the A peak related gene and performing GO and KEGG functional enrichment analysis;
the extracted sheep dorsum longus muscle tissue RNA is sequentially subjected to RNA specificity m 6 A antibody immunoprecipitation, library construction, and sequencing, including:
enrichment of extracted sheep dorsum longus muscle RNA by VAHTS mRNA capture beads and ZnCl addition 2 Incubation at 95℃for 5min-10min for RNA-specific m 6 A, immunoprecipitation of the antibody;
constructing a chain RNA sequencing library by using an Illunmia KC-DigitalTM chain mRNA library preparation kit;
the quality control filtering is performed on the raw data obtained by sequencing to obtain filtered data, and the filtered data is compared with the sheep reference genome data to obtain genome comparison position data, which comprises the following steps:
carrying out quality statistics on the raw data obtained by sequencing through FastQC software;
performing quality control by using NGSQC Toolkit to obtain the filtered data;
comparing the filtered data with the sheep reference genome data by using bowtie2 comparison software to obtain the genome comparison position data;
said aligning said genome to positional data by m 6 Authentication and annotation of A peak to obtain m 6 An apeak related gene comprising:
peak rolling scan using exomePeak, analyze m 6 The position of the A peak and the corresponding gene, and the threshold value is q value<0.05；
Using bedtools vs. m 6 Labeling the peak A;
the m is treated with deep tools 6 Analyzing the peak value distribution of the peak A;
the m is 6 The difference in peak a was determined by python script using a fisher test;
merging adjacent significant enrichment regions into one enrichment region as the final m 6 A peak；
Extraction of m Using Homer 6 The sequence of the interval in which A peak is located, for m 6 Scanning the common motif among the A peaks, and searching for a common motif region;
m between repeated samples in a group 6 A peak filters, retains overlay>50% of total m 6 A peak related gene.
2. The method for mining sheep skeletal muscle development related genes based on the MeRIP-Seq technique according to claim 1, wherein the said method is based on the said m 6 A peak-related genes give a difference m 6 A peak-related gene, and for the difference m 6 The A peak related gene is identified and GO and KEGG functional enrichment analysis is carried out, which comprises the following steps:
according to said m 6 A peak related gene, fold Change>2，P<0.05 is the screening difference m 6 A peak related gene standard, obtaining the difference m 6 A peak correlationA gene;
the difference m is treated with KOBASA 6 The A peak related gene was subjected to GO and KEGG functional enrichment analysis.
3. The method for mining sheep skeletal muscle development related genes based on the MeRIP-Seq technology according to claim 1, wherein sheep skeletal muscle m at different developmental stages 6 The motif of A corresponds to the motif RRACH.
4. The method for mining sheep skeletal muscle development related genes based on the MeRIP-Seq technology according to claim 1,
skeletal muscle difference m of sheep at different developmental stages 6 The GO analysis of A expressed peak annotated genes are enriched for protein binding, enzyme binding and developmental processes;
skeletal muscle difference m of sheep at different developmental stages 6 KEGG analysis of a gene annotated to express upregulated peak is enriched in circadian, hormone-regulated signaling pathways;
skeletal muscle difference m of sheep at different developmental stages 6 GO analysis of A expressed down-regulated peak annotated genes were enriched for tRNA metabolic processes, RNA methylase activity and ncRNA metabolic processes;
skeletal muscle difference m of sheep at different developmental stages 6 KEGG analysis of a genes annotated for peak down-regulated expression were enriched in PI3K-Akt signaling pathway, rap1 signaling pathway, foxO signaling pathway and cGMP-PKG signaling pathway.
5. The method for mining sheep skeletal muscle development related genes based on the MeRIP-Seq technology according to claim 1, wherein the low m involved in sheep skeletal muscle growth and development 6 The A methylation high expression genes are TGFB3, FGF16, FGFR1 and CSF1R.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310613009.9A CN116343929B (en) | 2023-05-29 | 2023-05-29 | Method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310613009.9A CN116343929B (en) | 2023-05-29 | 2023-05-29 | Method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116343929A CN116343929A (en) | 2023-06-27 |
| CN116343929B true CN116343929B (en) | 2023-09-08 |
Family
ID=86891535
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310613009.9A Active CN116343929B (en) | 2023-05-29 | 2023-05-29 | Method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116343929B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628766A (en) * | 2019-09-23 | 2019-12-31 | 中国农业科学院北京畜牧兽医研究所 | LncRNA coding gene related to sheep skeletal muscle development and application thereof |
| CN111261229A (en) * | 2020-01-17 | 2020-06-09 | 广州基迪奥生物科技有限公司 | Biological analysis process of MeRIP-seq high-throughput sequencing data |
| CN113462776A (en) * | 2021-06-25 | 2021-10-01 | 复旦大学附属肿瘤医院 | m6Application of A modification-related combined genome in prediction of immunotherapy efficacy of renal clear cell carcinoma patient |
| CN114480670A (en) * | 2022-01-26 | 2022-05-13 | 西北农林科技大学 | DNA methylase TET1 and m related to bovine skeletal muscle differentiation6Interaction identification and application of A methylase METTL3 |
| WO2022204362A2 (en) * | 2021-03-25 | 2022-09-29 | The Broad Institute, Inc. | Compositions and methods for treating a neurodegenerative or developmental disorder |
-
2023
- 2023-05-29 CN CN202310613009.9A patent/CN116343929B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628766A (en) * | 2019-09-23 | 2019-12-31 | 中国农业科学院北京畜牧兽医研究所 | LncRNA coding gene related to sheep skeletal muscle development and application thereof |
| CN111261229A (en) * | 2020-01-17 | 2020-06-09 | 广州基迪奥生物科技有限公司 | Biological analysis process of MeRIP-seq high-throughput sequencing data |
| WO2022204362A2 (en) * | 2021-03-25 | 2022-09-29 | The Broad Institute, Inc. | Compositions and methods for treating a neurodegenerative or developmental disorder |
| CN113462776A (en) * | 2021-06-25 | 2021-10-01 | 复旦大学附属肿瘤医院 | m6Application of A modification-related combined genome in prediction of immunotherapy efficacy of renal clear cell carcinoma patient |
| CN114480670A (en) * | 2022-01-26 | 2022-05-13 | 西北农林科技大学 | DNA methylase TET1 and m related to bovine skeletal muscle differentiation6Interaction identification and application of A methylase METTL3 |
Non-Patent Citations (1)
| Title |
|---|
| m6A甲基化酶相关基因在牛骨骼肌生成中的表达;杨昕冉 、马鑫浩 、杜嘉伟、昝林森;《中国农业科学》;165-178 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116343929A (en) | 2023-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Landscape and regulation of m6A and m6Am methylome across human and mouse tissues | |
| JP7072091B2 (en) | Methods and applications of gene fusion detection in cell-free DNA analysis | |
| Hezroni et al. | Principles of long noncoding RNA evolution derived from direct comparison of transcriptomes in 17 species | |
| CN110325650A (en) | Method and system for analyzing nucleic acid molecules | |
| Korpelainen et al. | RNA-seq data analysis: a practical approach | |
| CN105793689A (en) | Methods and systems for genotyping genetic samples | |
| CN113463202B (en) | Novel RNA high-throughput sequencing method, primer group and kit and application thereof | |
| US20180135120A1 (en) | Comprehensive methods for detecting genomic variations | |
| CN112863595A (en) | Method for excavating Tibetan sheep high-altitude hypoxia adaptability related gene based on MeRIP-Seq technology | |
| CN116343929B (en) | Method for excavating sheep skeletal muscle development related genes based on MeRIP-Seq technology and application | |
| CA3119980A1 (en) | Methods, compositions, and systems for improving recovery of nucleic acid molecules | |
| Eché et al. | A Bos taurus sequencing methods benchmark for assembly, haplotyping, and variant calling | |
| US20200131566A1 (en) | Methods, compositions and systems for calibrating epigenetic partitioning assays | |
| CN113122642A (en) | Method for assembling and annotating Hu sheep genome based on third-generation PacBio and Hi-C technology | |
| CN114746560A (en) | Methods, compositions, and systems for improved binding of methylated polynucleotides | |
| CN110684830A (en) | RNA analysis method for paraffin section tissue | |
| KR101967879B1 (en) | Method for measuring integrity of unique identifier in sequencing | |
| HR MAS et al. | From DNA to Data: Transcending Beyond the Double Helix and Demystifying the Genetic Alchemy of Life Through NGS to Empower Precision Medicine | |
| WO2024006361A1 (en) | Nucleic acid probes | |
| Liu et al. | When Livestock Genomes Meet Third-Generation Sequencing Technology: From Opportunities to Applications | |
| CN117965734A (en) | Gene marker for detecting hard fibroid, kit, detection method and application | |
| CN104561261A (en) | Method for designing gene chip for detecting lncRNA (long non-coding RNA) of milk cow breast tissues |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |