CN113444807B - Sheep fat-related circRNA and application thereof - Google Patents

Abstract

The invention relates to a circRNA related to sheep fat and application thereof. The research on the molecular mechanism of fat deposition of sheep can improve the quality of meat products so as to meet the requirements of consumers and avoid the waste caused by excessive conversion of feed into fat in the breeding process. In order to find out new circRNA related to subcutaneous fat of sheep, the inventor selects two representative sheep with larger difference in fat deposition as research objects based on a complete transcriptome sequencing technology, screens and identifies candidate genes related to fat metabolism, and verifies through a molecular experiment, so that the application lays a foundation for the breeding of sheep varieties.

Description

Sheep fat-related circRNA and application thereof

Technical Field

The invention relates to the technical field of agricultural genetic engineering, in particular to a circRNA related to sheep fat and application thereof.

Background

Lipid metabolism is mainly performed in animals by liver or adipose tissue, and is expressed in the form that fat is digested, absorbed, synthetically decomposed by the action of relevant enzymes, and mainly comprises the metabolism of 4 types of substances: phospholipids, triglycerides, cholesterol and proteins. At present, the living standard is greatly improved, and the requirements of people on the quality and quantity of meat are also improved, so that the research on the molecular mechanism of sheep fat deposition can improve the quality of meat products so as to meet the requirements of consumers, and the waste caused by excessive conversion of feed into fat in the culture process can be avoided.

circRNA is the first closed circular RNA molecule found in plant viruses and influenza viruses, generated by specific cleavage. As circRNA does not have a 5 'cap and 3' poly A structure, it is more stable than linear RNA because ribonuclease cannot degrade circRNA. Endogenous competitive circRNA can also affect miRNA activity, thereby regulating gene expression, presumably circRNA may regulate adipogenic differentiation via the above principles. The research finds that 31 circRNA molecules in a precursor adipocyte in-vitro differentiation system test can be obviously increased along with the fat differentiation process, and correlation analysis shows that the expression of the circRNA molecules can be related to the corresponding linear RNA expression state. Research on the subcutaneous adipose tissues of Laiwu pigs and white pigs revealed that circRNA _26852 and circRNA _11897 can target genes that are enriched in pathways related to lipid metabolism and disease. CircACC1 promotes fatty acid beta oxidation by promoting LO2 hepatocyte AMPK holoenzyme assembly, activity and stability. Therefore, it is speculated that circRNA is possibly involved in the regulation of lipid metabolism and can provide a potential target for lipid metabolism related diseases.

The research related to sheep fat is less at present, and in order to further discover new circRNA related to sheep subcutaneous fat, the inventor selects representative pluronic sheep (D) and small tailed han sheep (X) with larger difference in fat deposition as research objects based on a high-throughput sequencing technology, wherein the former belongs to meat and fat dual-purpose sheep, the fat hip is larger, the latter belongs to short-lean-tailed sheep, the subcutaneous fat is less, and key candidate genes related to fat metabolism and fat forming differentiation are screened and identified, so that a molecular mechanism related to fat deposition is explored.

Disclosure of Invention

The primary object of the invention is to provide a circRNA related to sheep fat, which is obtained by screening through whole transcriptome sequencing and using molecular experiment verification.

A circRNA, the sequence of which has more than 90 percent of sequence homology with SEQ ID NO. 1.

Preferably, the circRNA sequence has more than 95% sequence homology with SEQ ID NO. 1; more preferably, the long non-coding RNA sequence is SEQ ID NO. 1.

The second purpose of the invention is to provide the application of the circRNA in predicting or assisting mutton quality.

The third purpose of the invention is to provide the application of the circRNA in preparing a reagent for predicting or assisting mutton quality.

The fourth purpose of the invention is to provide the application of the circRNA in breeding sheep with different muscle qualities.

The meat performance refers to the performance in the aspects of meat yield, carcass composition, fat content in meat, meat grade, fat color, intramuscular fat content and the like.

The fifth purpose of the invention is to provide a method for detecting the fat content of sheep, which comprises the following steps: (1) selecting sheep adipose tissues; (2) detecting the expression level of the circRNA in the sample.

Preferably, the fat is subcutaneous fat or intramuscular fat

It is a sixth object of the present invention to provide a product comprising a reagent for detecting the above circRNA in a sample.

Preferably, the product comprises a chip, a kit or a nucleic acid membrane strip.

Preferably, the reagents comprise reagents for detecting the expression level of the circRNA in the sample by sequencing technology, nucleic acid hybridization technology or nucleic acid amplification technology.

Preferably, the expression level of the circRNA in the sample is detected by adopting a high-throughput sequencing technology, a probe hybridization technology, a gene chip technology or a fluorescent quantitative PCR technology.

Preferably, the reagent comprises a probe that specifically recognizes circRNA, or a primer that specifically amplifies circRNA.

Preferably, the primer sequence for specifically amplifying the circRNA is SEQ ID NO.2 and SEQ ID NO. 3.

Preferably, the sample is adipose tissue, and more preferably the fat is subcutaneous fat or intramuscular fat.

The seventh purpose of the invention is to provide the application of the product in predicting or assisting mutton quality.

The eighth purpose of the invention is to provide the application of the product in preparing an agent for predicting or assisting mutton quality.

The ninth purpose of the invention is to provide the application of the product in breeding sheep with different muscle qualities.

"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or an analog thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for example, a polynucleotide as defined herein includes, but is not limited to, single-and double-stranded DNA, DNA comprising single-and double-stranded regions, single-and double-stranded RNA, and RNA comprising single-and double-stranded regions, hybrid molecules comprising DNA and RNA, which may be single-stranded or, more typically, double-stranded or comprise single-and double-stranded regions. In addition, the term "polynucleotide" as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in such regions may be from the same molecule or from different molecules. The region may comprise the entire population of one or more molecules, but more typically is a region comprising only some molecules. One of the molecules of the triple-helical region is often an oligonucleotide. The term "polynucleotide" specifically includes cDNA.

Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. Modification of the nucleotide structure may be performed before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those having uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those having charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those having intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those having modified linkages (e.g., alpha anomeric nucleic acids), and unmodified forms of the polynucleotide. In addition, any hydroxyl group typically present in sugars can be replaced with, for example, a phosphonate group, a phosphate group, protected with a standard protecting group, or activated to make additional linkages to additional nucleotides, or can be conjugated to a solid or semi-solid support. The 5 'and 3' terminal OH groups may be phosphorylated or substituted with amines or organic capping group modules of 1-20 carbon atoms. Other hydroxyl groups can also be derivatized to standard protecting groups. Polynucleotides may also contain analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl-, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methylribonucleosides. One or more phosphodiester linkages may be replaced with alternative linking groups. Not all linkages in a polynucleotide need be identical. A polynucleotide may contain one or more different types of modifications described herein and/or multiple modifications of the same type. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "oligonucleotide" generally refers to a short, single-stranded polynucleotide that is less than about 250 nucleotides in length, although this is not required. The oligonucleotide may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides is equally and fully applicable to oligonucleotides.

"primer" as used herein refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of the complementary nucleic acid, typically by providing a free 3' -OH group.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. These techniques include, but are not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Illustrative, non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

The present invention can amplify nucleic acids (e.g., ncRNA) prior to or simultaneously with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using RNA replicase (commonly known as Q.beta.replicase), transcription-based amplification methods, and self-sustained sequence amplification.

In the present invention, the product comprises a reagent for detecting CIRCRNA, and one or more substances selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

The kit of the invention can be accompanied with instructions for using the kit, wherein the instructions describe how to use the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme

The invention has the advantages and beneficial effects that: (1) the sequence shown as SEQ ID NO: 1 and the circRNA related to the performance of the sheep meat; (2) in actual production, the meat performance of sheep can be improved and the meat feeling can be improved by regulating and controlling the expression of circRNA, so that the invention provides theoretical basis and technical revelation for breeding in animal husbandry.

Drawings

Figure 1 is an adipose tissue differential expression circRNA GO annotation;

FIG. 2 is an adipose tissue differential expression circRNA KEGG pathway enrichment assay;

FIG. 3 shows the results of the validation of the circRNA gene differential expression qRT-PCR.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

EXAMPLE 1 Collection agent RNA extraction of samples

1.1 Collection of samples

The test was conducted on Doudang sheep (D) and small tailed han sheep (X) with differences in fat deposition. The selected sheep were all adult females, and the individuals were well-conditioned, healthy and disease-free, of similar body weight (about 50kg), with three biological replicates per group.

1.2 extraction and quality detection of sample RNA

Equal amounts of adipose tissue were removed from each sample for total RNA extraction, ensuring that the entire procedure was performed in a sterile environment, as follows:

(1) respectively grinding adipose tissues in a mortar added with liquid nitrogen;

(2) placing the ground tissue into a centrifuge tube added with Trizol reagent for standing;

(3) adding chloroform, mixing and standing for 10 min at room temperature;

(4) placing the mixture into a centrifuge, and centrifuging the mixture for 15 minutes at the rotation speed of 12000 under the condition of 4 ℃;

(5) putting the colorless liquid at the uppermost layer into an RNase free tube, precipitating RNA by using isopropanol with the same amount, uniformly mixing at room temperature and standing for 10 minutes;

(6) centrifuging again for 15 minutes at the temperature of 4 ℃ and the rotating speed of 12000;

(7) removing supernatant, adding 75% ethanol into the precipitate, mixing, and centrifuging under the above conditions;

(8) removing supernatant, and continuously adding ethanol to wash RNA2-3 times;

(9) removing ethanol, and drying the precipitate for 5-10 min;

(10) finally, the precipitate is treated with RNA enzyme-free water.

To ensure the purity and integrity of total RNA, the total RNA was subjected to quality testing:

the requirement of library construction and sequencing is that the Total amount of RNA is not less than 2ug, a TruSeqTM Stranded Total RNA Kit is adopted for the library construction of longRNA (mRNA, lncRNA and circRNA), the Total amount of RNA is 2 mug, the concentration is more than 100 ng/muL, and the OD260/280 range is 1.8-2.2.

Total RNA is extracted from 6 subcutaneous adipose tissues of the Wulang sheep and the small-tailed han sheep respectively and quality detection is carried out, and the result shows that the brightness of a 28S strip is obviously higher than that of 18S strip; the RNA concentration of each sample is higher than 200 ng/mu l; RIN values are all larger than 8, OD260/280 is larger than or equal to 1.8, OD260/230 is larger than or equal to 1.0, and the result shows that RNA is not polluted by carbohydrate, protein and other impurities, the integrity is good, and the RNA can be continuously applied in subsequent tests.

Example 2 sequencing and data analysis

Sequencing was performed using the Illumina NovaSeq 6000 sequencing platform. The circRNA is in a closed ring shape, is formed by reversely connecting exon splicing acceptors and donor sites which are respectively positioned at the upstream and the downstream, belongs to a non-coding RNA molecule, has no influence on the non-coding RNA molecule by RNA exonuclease, is not easy to degrade, and is more stable. Linear RNA fragments can be aligned by linear alignment software, but Reads in trans-splicing positions of circular RNA cannot be aligned directly to the genome. circRNA prediction was performed using correlation software (CIRI2+ find _ circ collection) based on Back spot connections (BSJ) reads and compared to the database circBase (animals).

Classification is based on the starting and ending points of circRNA on the genome. The first type of termination point and the initiation point are both located in the exonic region of the gene and are exon circRNA. One end of the second type of initiation or termination point is located in the intron region of the gene and is intron circRNA. A third class of circRNAs is classified as endogenous circRNAs when either the start or stop point is located in the intergenic region or when both start and stop points are located in the exon regions of a gene but the two exons are not in the same gene.

And analyzing the difference of sample expression. Using rpm (reads per millon reads) as a quantitative index, the calculation formula is:

RPM＝Total exon reads/Mapped reads(Millions)

wherein, Total exon reads represent all reads from a certain sample Mapping to a specific gene exon; mapped reads represent the total amount of all reads for a sample. 18947 circRNAs are obtained in subcutaneous adipose tissues of Dolang sheep and small tailed Han sheep, differential expression analysis is carried out on the data, (screening is carried out by taking the standard of | Fold Change | ≧ 2.0 and Pvalue < 0.05), 141 circRNAs with differential expression coexist in subcutaneous adipose tissues of two varieties of sheep, wherein 61 differential genes are up-regulated and 80 are down-regulated. The circRNA-NC-040258.1-1701203-1708891 with remarkable differential expression is found for the first time in the application as a candidate molecule for the next time.

To understand the function of differentially expressed genes and gene products, three-way annotation analysis was performed on differentially expressed genes using GO annotation, including biological processes, cellular components, and molecular functions. The source genes of the different circRNAs are analyzed, and the enrichment is found to be 379 entries in total, and 99 entries are screened under the condition that Pvalue is less than 0.01. As shown in fig. 1, the differentially expressed circRNA-derived gene is mainly enriched in terms of translation inhibitor activity (translational activity), leptin receptor binding (leptin receptor binding), triphosphatase activity (triphosphatase activity) and the like in terms of molecular functions; in the cellular fraction, it is mainly enriched in ribonucleoprotein granules (ribonucleoprotein granules), fibrillar collagen trimers (fibrillar collagen trimers), nucleotide-activated protein kinase complexes (nucleotid-activated protein kinase complex), cytoplasmic ribonucleoprotein granules (cytoplasmic ribonucleoprotein granules); in biological processes, protein activation cascade (protein activation cascade), plasma membrane bound cell projection assembly (plasma membrane bound cell projection assembly), vesicle cytoskeletal transport (vesicle cytoskeletal transfer), and regulation of TOR signaling (regulation of TOR signaling) are among the processes. Through GO function research, the source gene of the differential expression circRNA is found to be involved in fat development by regulating and controlling leptin receptor binding enzyme, triphosphatase and other enzyme activities related to fat deposition.

In subcutaneous adipose tissues of a pluronic sheep and a small-tailed han sheep, the source genes of the circRNA are enriched into 110 passages (fig. 2, screening is carried out under the condition that Pvalue is less than 0.05, 3 remarkable enrichment passages are obtained in total), Protein digestion and absorption (Protein differentiation and absorption), Glucagon signaling (Glucagon signaling pathway) and lipolysis (Regulation of lipolysis in adipocytes) are obtained, KEGG passage analysis shows that the source genes of the circRNA are differentially expressed in the subcutaneous adipose tissues of the pluronic sheep and the small-tailed han sheep and participate in the passages related to fat metabolism, and the differential genes enriched in the passages can play an important role in regulating and controlling the subcutaneous fat deposition of the two varieties.

Example 3 real-time fluorescent quantitative PCR assay validation

In the experiment, 20 pluronic sheep and small-tailed han sheep are respectively collected, PCR detection is carried out on the circRNA with differential expression between the two groups, each gene is repeated for 3 times, and the differential expression condition of the NC-040258.1-1701203-1708891 gene is verified.

The main method comprises the following steps:

reverse transcription was performed using the circRNA cDNA Synthesis Kit (GenePool, Cat # GPQ1805) in the following reaction scheme: 2 × FastSYBR texture, 10 μ l; 0.4. mu.l each of the downstream primer (10uM) and the upstream primer (10 uM); template, 2. mu.l; distilled water was sterilized to 20. mu.l. The primer information is shown in the table.

TABLE 1 PCR reaction results

After the reaction is finished, data are derived, using 2^-ΔΔCtCalculating the relative expression of the sample by the method, analyzing the relative expression by t-test statistics, and displaying the data as the average +/-standard deviation (Mean +/-R)SD), P <0.05 is significant. Analysis of PCR data and results, using 2^-△△CtThe relative expression of the circRNA gene between the samples was calculated and the data are expressed as Mean. + -. standard deviation (Mean. + -. SD), P<0.05 indicated that the gene differed significantly between the two groups. The results show that the NC-040258.1-1701203-1708891 gene is significantly up-regulated in subcutaneous adipose tissue of Lance-warrior sheep.

The method obtains the circRNA gene closely related to fat deposition through functional annotation and enrichment analysis screening, and the conclusion is further verified through a fluorescent quantitative PCR experiment result.

Sequence listing

<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences

<120> sheep fat-related circRNA and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

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<211> 7688

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gcagtgaaac ctcattcttt tttgaagctc gaagtaaaac tgcttgcaag catctttgga 60

aatgtagtgt agaatatcac acatttttta ggtatgttcc tttgcctaat aaatgaaaat 120

tgcgtttgct tttataacaa gaattctatt cttagaagaa atgtataagt gtttctcttt 180

tcttaacttc tttacatgca catgtatgta gatgttttgt ttgtttgttt gctcatgcta 240

taaggaagga tcaaggatga ttgcttttgg ggtttatgta aaagtatagc tttggtattg 300

gtgggagtta agtgttctgc tgtgaacaca ctaattgaaa tgcctagcct cgaaatatgg 360

aatgggtggc tgggtatgta aagcttggtc atgtaaattt tgtaggtaat aaggcaggca 420

gtaggcagac agtcgccaat ggaaacagta aagagaggac agggactgcg ctgtgggacc 480

cttccagcgc ttagaggacg ggagcgtggg gagtgggaag taagggcggc agatgctgtg 540

acgagtgagg agctggcgtc tgccttgtgt gctcctggga ggtcgcaaag atgcaggcag 600

agcactgacc gttgggttgt gtgggtactg cttgtttgac attcttatgg tgtaaagatt 660

aaattttatc aggtaatgtt atacattaaa ctgtttttgg ttttcaaatt tagattaatc 720

cttccccttt ttcctttttg atttacacag aacatttttt ccaaatagta tgacgctaga 780

ggagtagtat tataaaacga tccacatctt ttgaggagtt tgtatccaga aagggatgta 840

gcccagaatg tctgagacat tacctctccg tcagtggggc ggggccaaga aactccactc 900

agagctgtgt gttgaatgta ccatagaatg gaatatgagg aagagtgaac catagcatta 960

gatatgatga actgccatga gaaccttggc ttgggttaat caacacagtg gcaaagttcc 1020

caagagaatt taagtagaga aattaaattt attcacaaca aaatcttcct tataagggtg 1080

atgatatatt taatagtctt caggaagtat gagtattttg atatcatact tgtataaaat 1140

agtagaatgc ggtggttgtt ttaagtatta gcttgaaagt aatcaacgat tattttttgc 1200

caatatgtag actgagtaaa atatttcaac tatgtgttaa gcagtattgc actattgcat 1260

atttctacgt agtttttgct cgtgttacac aagtatattt gtaagtcttt ttctaatttt 1320

cttttacaaa ttcattagaa tgccagaaaa tgaatcaaat tcattgtcaa gaaaactcag 1380

caagtttgga tccatgagtt ataagcaccg gtataggtaa atggcattct tttcctttgg 1440

ggtgtgtgga tgtgtgtatg aatgcatatt taatctaacc atccagatgc atgcatgcat 1500

attaaagttt gtgatctaat ttttttctga tttttttcat aatgccagtt attttgatag 1560

gcaataacag tttattaaac tttcttgaca aaaatgattt tctaggttta atgatattta 1620

aagagtacat tagcttgatt aagtatagag tttttagaat gtagtagagc ttactatctt 1680

cagaaattca ttttggtcca attatcagtt atctccaatt atgtggtgat tatgaaacag 1740

agtagatatt ctgaatagta gtctattggc attcttagga tattcagtta attctctgtt 1800

gcagcatcat gggtcaaatg ttttctgtag tataatgctt acaacatagt aatgctagca 1860

agtatatgtt aactttatgg agcagtaccc agtgctatat attgtgctaa atgtgtccca 1920

atatcgcttc tttaatcatt gcagtcttat tgatgttatc ctcataccat gtgaggaaac 1980

tgaggctcag agacaagtca cttttcagaa aaaccaacca ggaaatgtcg atgttgggcc 2040

cactgtctgg cagtctaact ccagacagag cctgtgtctt tactggttag tcagactgtt 2100

gcatgttgcg aggaaagctt gattgtcaag cagccaggca ggtgcttgtg aaaatggaaa 2160

gtttctatgt tttgaaaaat aatttgtttt gtttcaattc attctgttaa ctggagaacc 2220

tgtgatattg aaggcaggat aataggtgcc cagagataga aagaataaga tttcaccacc 2280

tttgccttcg gcttccctag tggctcagac gtaaagaatc tgcctgcaat gtgggagacc 2340

tggatttgat acctgggttg ggaagaaccc ctggagaagg gcatggcaac ccactccagt 2400

attcttgcct ggagaatccc atggacagag actgtagact ggcgggctac agtccatggg 2460

gtcgcaaaga gtcggacacg actgagcaac taacacctct gccttcagag agctacagta 2520

ttattcagtg agcagtgata cagcagtgag atggtcccag gccctataaa actgaagcag 2580

ggtgcagaaa tggcactttg gaggcaggac actgttctgt cagtctattt gagatatctc 2640

caagtgaaat gcttacttag gagacttaaa aatcaaacca ggtcaagcgt gtgcaagaca 2700

ataacatgcc agattttaat tcctttcttt tataaaccac tttttaaaat ctcactttga 2760

aaccagggat aatcagtaaa ggagtgatta atattaaatg ttcagtacat taatctttac 2820

atattcactt ggatgtttcc tcatttgctg acttaacact taagtattat tatattatag 2880

atgttcttgg tgtttgtagg aaagtgttta ttgaaatcca aaaatttaaa ctttgttatc 2940

aaggttttcc cctagatagt gatataatta caggaattta aggagagatt atttactttt 3000

atattaagga aaacagctca tagtgagtat ttgggctgtt cttcccactg ggacgtaaca 3060

catttcaagg gttatagagt gaatgtgtaa acctgtctct ttgtcactga aatgaagcta 3120

gtgaaaaagc atctgtctta atattcataa tgcaaaattt ttaattatgc tgtttaacat 3180

acactgtagc tggtttctac aaatttacca tacctgactt ttcaaattta ttgaaatgca 3240

aatgctgtca ctctgggaat tattattagg aagttattgg attttgcata ttaagtataa 3300

tttcctccac aaacgtaatt aaaggtaacc tttgcaaatt ggaagctctg ctaaatctgt 3360

tctaattttg tgggtggtgt taactgctta gtaaatctta atgaaattcg agtctcagtg 3420

tctgtacgta ggaagcacta acattatgca gttcctggaa gaaggatgtt tcatcccact 3480

acccagagcc aggcttaggc agggatgggt ctctgactac tgggtggtca ccaggacatg 3540

actgttaatt gtcaagagca ataacagcca atctttaaaa aacatcgtat gcctgttcct 3600

ttattttgag acgtgctgga gacaacctgg aaggatggaa gaactctgga aacagaaagc 3660

tccttccttt accctgtagg cttttagagc gtgggagata gtgaacggca taaccagtgg 3720

ctgtggtgtg tgagtgtggt ggtctgtgtg tataactctt aatgtgttta catgtgttac 3780

gtgtgtgtag gagtgtgtga tagagagaga cagttctagt ctttactttc tcttttgcat 3840

cttagggctt gcagaatacc aaggagattc caacatcttt gagaaaaaac tagtcattac 3900

tttagaggag aaaacttgat tttagtggta tgtgacaggc aatcctttca gaaattcaaa 3960

attaaatttt tgggaaaaga tagagctgaa atgtgacagt caaagtcatt tattgcttta 4020

ttatcagata ccagtaatct gaaatagaga ctgattaagt ctttggttaa atattttgtt 4080

atacactgct ctctcttcct ctgagcccct taaaaactaa ccttgcctct acctgttgtt 4140

aagtttgtag aggactcctg gtttggatga atgaatgagt cgtgtagagc ttatactcta 4200

gaaatgagac ttcgacctga gaaccacctc ccgcccgcag atccccgata gccttcttgc 4260

cacatttctt gaagtgtgca agagatccag tcctcctgag aacttacttt gtctcaagtg 4320

ctttcatgtg tgttacttgg ttaaatacat tcagtgaccc atcaaagggg ccttatcctc 4380

atttgtacag acaagatatt tgagcaccgc cccccccccc cccccccccc cccccccccg 4440

acgccaagtc taaggtaatt gaactgaagg cctttagcca gtaagcagca gagttgagat 4500

ctgaacccag gtgtctgtcc tcttccctct acccaaccta tggctgtttg tagcctcact 4560

ttggggctga agaagggctg ctggcggggc ctcccagcat ggaatgggag tgtgtgccac 4620

agggcgaggg gtgggcacgt gggtgacatg ggggcaaagc tctatcctgc cgctgcggag 4680

agccactagg cgggcaggga cgcttacacc aacatatcct gccgctgcgg agagccacta 4740

gggcaggcag ggacgcttac accagcactg agaatctctg catttgccct gtgcttttct 4800

gttacaagga aggactaaga acaccataca ggagtgtttc tgctagggaa taataggagc 4860

aatagaaata cagacgagga aaataaggtt ctaggagatt aaaaaaataa cctactgaag 4920

accacgcgtt tctgctagag ctgagctttg agccaggctt gtagctttaa agtacatggc 4980

tgtgtgcgct gttccttctc ctgggctgcc aggctgtgcc accctggcca gctggaggat 5040

agaaagctgc ttccggtcct gggtcctgtg cagcctctgc caggtcagct tatgtccctg 5100

gacttcagtt gtattagtct tgtgatatga ttttctcatt tcttctttat tcctgagaaa 5160

caagccatga ggaaaaattg aataataaaa taaaatctca ttatcaaatg cctcagtagt 5220

ccacatatta ggttatgttt ttaggccaaa aattttttcc tccatgcaat gaagtacaga 5280

ctatccaagt cttagattaa gtctgttgca aggagatctt atcttcatac ttcaaataat 5340

ttataataat ggaattttgc tgtctgtata gaattttaaa aagattttca ggggagagat 5400

gttgctattt gtattacctt attcactacc tttagaatag ctaaaactgt tgaaagttct 5460

caaattaata atttttaaaa tgtgtttatt tgtatttagt tataattatg taacatttta 5520

tgtgcttggc attgttctgg aaaatttaga aacattcatg cttaacattc ataataagcc 5580

tataaagtgg tcattggtat tattgttgct ttagtaatga ggaagccgag ctacagacag 5640

tttgagtaat gtttcccgga gtcagagagc taggctatga catagctgga atttgaaccc 5700

agtgtccaga gcttcttatt aacaatatat taatgtgtga aaacagcaaa atgaaagttg 5760

ataaatgaca attttctttt catgttatta gatttctata tggtacattt tcactatata 5820

tgaggggaag tagttacatt tcaagtggaa gcaagcctgt cactctgtgc cccccagccc 5880

cctgccctcc ccctggtttt catgaagtct tgaatgtgga gtatgtacac tcacggatag 5940

aaactgtcgt tttcattcca catagtaagt cgtatcccca gttagcccga tgagaaatag 6000

cttgttatga atgtttcagg cctctgtttc ttgtaaaaat gaatggggta gtaagcaagg 6060

agggggagtt tccacagaag tatgtaggag gtaaattcgg ctttttccgt ctcatttgat 6120

ctcttttggg gaaagagcca ccagcacgta tcctgtgctg ctgctgctaa ggcacctcag 6180

tcgtatccga ctgcgtgccg ctccacagac ggcagcccag caggctcccc cggccctggg 6240

actctgcagg caggagcacc ggagtgggct gccatgtgtc ctgtaaatgg agggcaaata 6300

ggttgaggcc attaaaagac ctttttccca ttagatggcg cttcctatag tgaatcaaag 6360

tagtcactga gaaattcatt ttctctgtac ctaaatgaca ctttccttaa aatccaagag 6420

tcatttattg gttcagattc agcagctgtg gaataaattt ttagtagaat gaatgttgac 6480

tgaaattcag caaattaagt tacatggttc ttgagaagtt tagtttctcc tcagggaaag 6540

ctcaccctgt tgcctcatag actcataagg aagtgctgct gtgccggggg attctggctg 6600

ctctgaatct tcctgggtac ttgctggcac aggtggggtg ggcggaatca ggggctgatt 6660

gtgtcacagg gttcagagta cagtctcgcc aagtggccca ctggcagagg ctctgtgact 6720

tgccttgtga gtacagcacg aggacatgtc gactcatgtg gaatctttgc cctcagtact 6780

tcagcccagc acggtttcat taccaagttg tggagctgac aacatgagca tcctcagttt 6840

ctagcctatc attctactta aatccaaacc tgattcctcc tttaataaat tttttcttgg 6900

ttctctctct gaaaaatctt ccttatgtca gctgagggga aaaaaaaagt gcaacatgag 6960

agctgtgagt caggctttat tggtgcttgt agagagagga ctagaggctg agagatggtc 7020

cttcagaggg ctctgaggac ctgcccctca gggagtgtgt gcagtcaaac acacgcctct 7080

gcaggaggtt gctgccagtc gccaggagca gatgtctcca ttaatgactg cagtgctttt 7140

ctagtaggag aagatgcaag agaccaagct cgtaaaatat tctgaaaacg tctatctgaa 7200

gccctgtttt gccacttttt cccagagcac agagtgcctc agtcccaatc tctgtcccga 7260

atccctttca gagtatgtcg aaggtcagca acttagtggc tagtggcttc attcttgcag 7320

aaccagatga tgggtgacat tcttcagctg gcacttatat ctcaaccaaa cctttcctgt 7380

tggactttgt tacttccttg cttaacactg caatgagaat catttgaagc tcctatataa 7440

tctgcgtgag tctggtttgt ttttttcttt ctctccatac actgcagata tattctttat 7500

gagctgtgct ttaattttgc ggctgtgtgc tcttacctgt gttactcaaa atgttgtatt 7560

ttcaagtggc aggacagcac tacagatgag tcgagatctt tctattcaac ttccccggcc 7620

tgatcagaat gtgtcaagaa gtcgaagcaa gacttaccct aagagaatag cacaaacaca 7680

gccagctg 7688

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

taccctaaga gaatagcaca aacac 25

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cactacattt ccaaagatgc ttgc 24

Claims (12)

1. A circRNA having the sequence SEQ ID NO. 1.

2. Use of the circRNA of claim 1 for predicting subcutaneous fat content in sheep.

3. A method for detecting sheep fat content, comprising: (1) selecting sheep adipose tissues; (2) detecting the expression level of the circRNA of claim 1 in the sample.

4. The method of claim 3, wherein the fat is subcutaneous fat or intramuscular fat.

5. Use of a product comprising a reagent for detecting the circRNA of claim 1 in a sample for predicting subcutaneous fat content in sheep.

6. The use of claim 5, wherein the product comprises a chip, a kit, or a nucleic acid membrane strip.

7. The use according to claim 5, wherein the reagents comprise reagents for detecting the expression level of circRNA in the sample using sequencing, nucleic acid hybridization or nucleic acid amplification techniques.

8. The use according to claim 5, wherein the expression level of the circRNA in the sample is detected by high throughput sequencing technology, probe hybridization technology, gene chip technology or fluorescent quantitative PCR technology.

9. The use according to claim 5, wherein the reagent comprises a probe that specifically recognizes circRNA, or a primer that specifically amplifies circRNA.

10. The use according to claim 9, wherein the primer sequences for the specific amplification of circRNA are SEQ ID No.2 and SEQ ID No. 3.

11. Use according to claim 5, wherein the sample is adipose tissue.

12. Use according to claim 11, wherein the adipose tissue is subcutaneous adipose tissue.

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