CN113604578B - A kind of miRNA related to sheep fat and its application - Google Patents

Abstract

The invention relates to a sheep fat-related miRNA and application thereof. The research takes subcutaneous adipose tissues of Dulang sheep and small-tailed Han sheep as research objects, uses an RNA-seq technology to carry out sequencing analysis on genes and uses a molecular experiment to verify, and discovers a new miRNA related to sheep fat. The invention provides a basis for cultivating high-quality meat sheep and the like, and has good application value.

Description

A kind of miRNA related to sheep fat and its application

technical field

The invention belongs to the technical field of agricultural genetic engineering, in particular to a miRNA related to sheep fat and its application.

Background technique

miRNAs are short-chain endogenous non-coding RNAs, which are highly conserved and widely distributed in animals, plants and viruses. In many biological processes, miRNAs participate in the post-transcriptional regulation of genes, hindering mRNA translation or degrading mRNAs by base-pairing with the 3'UTR of complementary mRNAs. miRNAs have both promoting and inhibiting effects on adipocyte differentiation. miR-143 is the first miRNA found to play a regulatory role in adipogenic differentiation, and promotes adipogenic differentiation by inhibiting the expression of MAP2K5. The role of miR-21 in the differentiation process of 3T3-L1 adipocytes can significantly promote the differentiation of preadipocytes and increase the expression of adiponectin. LPL is a direct target gene of miR-152 during preadipocyte differentiation. miR-152 can inhibit the proliferation of 3T3-L1 preadipocytes and promote the differentiation of 3T3-L1 preadipocytes by negatively regulating LPL.

Different breeds of sheep have different fat deposition capacity. Duolang sheep and Small-tailed Han sheep are both high-quality local sheep breeds in my country, but Duolang sheep have stronger fat deposition ability than Small-tailed Han sheep, and belong to both meat and fat sheep, while Xiao-tailed Han sheep are short and thin-tailed sheep. Subcutaneous fat is less, and the difference between the two provides a good material for us to study fat deposition in sheep.

In order to explore the molecular mechanism of fat development, this study took the subcutaneous adipose tissue of Duolang sheep and Xiaoweihan sheep as the research objects, and used RNA-seq technology to sequence and analyze the genes, in order to cultivate high-quality meat sheep and prevent and treat obesity and lipid metabolism. Syndrome and other diseases provide the basis.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to provide a sheep fat-related miRNA, which is obtained by screening through sequencing and bioinformatics analysis and verified by molecular experiments.

A miRNA related to sheep fat, the miRNA is NC_040278.1_37602, and the sequence has more than 90% sequence homology with SEQ ID NO.1.

Preferably, the NC_040278.1_37602 sequence has more than 95% sequence homology with SEQ ID NO.1; more preferably, the miRNA sequence is SEQ ID NO.1.

Preferably, the fat is subcutaneous fat or intramuscular fat.

A pre-miRNA related to sheep fat, the preRNA sequence has more than 90% sequence homology with SEQ ID NO.2; preferably, the pre-miRNA sequence is SEQ ID NO.2.

The second object of the present invention is to provide a reagent for detecting properties related to sheep fat or meat.

The reagent detects the expression level of NC_040278.1_37602 and/or its target genes in the sample through sequencing technology, nucleic acid hybridization technology or nucleic acid amplification technology.

Preferably, the target gene of NC_040278.1_37602 is one or more of TBC1D19, PDE3B, CLIC4, PSD3, VAV3, TASOR2, SPATA6, and PTGS2 genes. More preferably, the target gene is PTGS2 gene.

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

Preferably, the fat is subcutaneous fat or intramuscular fat.

The third object of the present invention is to provide a kit for detecting properties related to sheep fat or meat.

The kit contains DNA chips, oligonucleotide chips, probes or primers necessary for DNA microarray, oligonucleotide microarray, Northern blotting, RNase protection assay and reverse transcription polymerase chain reaction, to The expression levels of NC_040278.1_37602 and/or its target genes were detected.

Preferably, the kit includes reverse transcription primers and/or amplification primers used in QPCR experiments, and the reverse transcription primers are Oligo(dT)-specific RT primers.

Preferably, the kit contains primer pairs for nucleic acid amplification to detect the expression level of the NC_040278.1_37602 gene.

The fourth object of the present invention is to provide the application of NC_040278.1_37602 and/or its target gene for preparing detection reagents related to sheep fat or meat performance.

Preferably, the expression level of NC_040278.1_37602 and/or its target genes in the sample is detected by sequencing technology, nucleic acid hybridization technology or nucleic acid amplification technology.

Preferably, the nucleic acid amplification technique uses specific primers to amplify NC_040278.1_37602; nucleic acid hybridization includes a probe that hybridizes with the nucleic acid sequence of NC_040278.1_37602.

Preferably, the nucleic acid amplification technique uses a pair of specific primers to amplify the NC_040278.1_37602 target gene; nucleic acid hybridization includes a probe that hybridizes with the nucleic acid sequence of the NC_040278.1_37602 target gene.

Preferably, the primer sequence used for nucleic acid amplification of NC_040278.1_37602 is SEQ ID NO.2.

Preferably, the sequences of the primer pair used for nucleic acid amplification of the NC_040278.1_37602 target gene are SEQ ID NO.3 and SEQ ID NO.4.

Preferably, the sample is tissue. More preferably, the sample is adipose tissue.

The fifth object of the present invention is the application of the above-mentioned miRNAs, pre-miRNAs, reagents and kits in the detection of products related to sheep fat or meat performance.

The sixth object of the present invention is the application of the above-mentioned miRNA, pre-miRNA, reagent and kit in breeding sheep for meat.

"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or may be incorporated into polymers by DNA or RNA polymerases, or by synthetic reactions any substrate in the substance. Thus, for example, polynucleotides as defined herein include, but are not limited to, single- and double-stranded DNA, DNA comprising single- and double-stranded regions, single- and double-stranded RNA, and single- and double-stranded regions RNA, a hybrid molecule comprising DNA and RNA, which may be single-stranded, or more typically double-stranded, or contain both single- and double-stranded regions. Additionally, 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 can be from the same molecule or from different molecules. The region may comprise the entirety of one or more molecules, but more typically will comprise only a region of some molecules. One of the molecules of the triple helix region is often an oligonucleotide. The term "polynucleotide" specifically includes cDNA.

Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure can be performed before or after assembly of the polymer. Nucleotide sequences can be interrupted by non-nucleotide components. Polynucleotides can be further modified after synthesis, such as by conjugation to labels. Other types of modifications include, for example, "caps", replacement of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as, for example, with uncharged linkages (e.g. methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and modifications with charged linkages (eg phosphorothioate, phosphorodithioate, etc.) containing pendant moiety such as eg proteins (eg nucleases, Modification of toxins, antibodies, signal peptides, poly-L-lysine, etc.), modification with intercalating agents (eg, acridine, psoralen, etc.), containing chelating agents (eg, metals, radiometals, boron, oxidizing metals) etc.), modifications containing alkylating agents, modifications with modified linkages (eg, alpha anomeric nucleic acids), and unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present in carbohydrates can be replaced with, for example, phosphonic acid groups, phosphate groups, protected with standard protecting groups, or activated to prepare additional linkages to other nucleotides, or can be Conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or replaced with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. Polynucleotides may also contain analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-oxo-methyl-, 2'-oxo-allyl-, 2'-fluoro- or 2'-oxo-methyl-, 2'-oxo-allyl-, 2'-fluoro- or '-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedum heptulose, Acyclic analogs and abasic nucleoside analogs such as methyl ribonucleosides. One or more phosphodiester linkages can be replaced with alternative linking groups. Not all linkages in a polynucleotide need to 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. Oligonucleotides can be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

A "primer" as used herein refers to a single-stranded polynucleotide capable of hybridizing to nucleic acid and permitting polymerization of complementary nucleic acid (generally by providing a free 3'-OH group).

The present invention can utilize any method known in the art to measure gene expression. As will be understood by those skilled in the art, the means of measuring gene expression is not an important aspect of the present invention. These techniques include, but are not limited to: nucleic acid sequencing, nucleic acid hybridization, 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 since RNA is less stable in cells and more susceptible to nuclease attack in experiments, RNA is typically reverse transcribed into DNA prior to sequencing.

Another illustrative non-limiting example of nucleic acid sequencing technology includes next-generation sequencing (deep sequencing/high-throughput sequencing), a single-molecule cluster-based sequencing-by-synthesis technology based on a proprietary reversible The principle of terminating chemical reactions. During sequencing, random fragments of genomic DNA are attached to an optically transparent glass surface. After extension and bridge amplification, these DNA fragments form hundreds of millions of clusters on the glass surface, each cluster has thousands of copies of the same template Then, using four special deoxyribonucleotides with fluorescent groups, the template DNA to be tested is sequenced by reversible sequencing-by-synthesis 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 method that uses labeled complementary DNA or RNA strands as probes to localize a section or section of tissue (in situ) or, if the tissue is small enough, whole tissue (whole tissue-embedded ISH) for specificity Hybridization of DNA or RNA sequences. DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and localize mRNAs and other transcripts (eg, ncRNAs) within tissue sections or whole tissue embeddings. Sample cells and tissues are typically processed to immobilize target transcripts in situ and increase probe access. The probes hybridize to the target sequence at high temperature, and the excess probes are washed away. Localization and quantification of probes labeled with radioactive, fluorescent, or antigen-labeled bases in tissues are 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 (eg, ncRNAs) prior to or concurrently 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 some amplification techniques (eg, PCR) require reverse transcription of RNA into DNA prior to amplification (eg, RT-PCR), while other amplification techniques amplify RNA directly (eg, RT-PCR). For example, 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 (in basic Autocatalytically synthesizes multiple copies of a target nucleic acid sequence under conditions of constant temperature, ionic strength, and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; the ligase chain reaction of LCR uses a Two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of nucleic acid; other amplification methods include, for example: nucleic acid sequence-based amplification commonly referred to as NASBA; amplification of probes using RNA replicase (commonly referred to as Qβ replicase); Amplification of the needle molecule itself; transcription-based amplification methods; and self-sustaining sequence amplification.

In the present invention, the kit includes reagents for detecting NC_040278.1_37602, and one or more substances selected from the group consisting of containers, instructions for use, positive controls, negative controls, buffers, auxiliary agents or solvents.

The kit of the present invention may also be accompanied by an instruction manual for the kit, which describes how to use the kit for detection, and how to use the detection results to judge tumor development and select a treatment plan

Advantages and beneficial effects of the present invention: (1) The miRNA with the sequence shown in SEQ ID NO: 1 and its correlation with the performance of sheep meat were discovered for the first time; (2) In actual production, the expression of related genes can be regulated by miRNA , thereby improving the meat performance of sheep and improving the meat feeling, so the invention provides theoretical basis and technical inspiration for animal husbandry production.

Description of drawings

Figure 1 is a graph of GO analysis of differentially expressed miRNA target genes in adipose tissue;

Figure 2 shows the enrichment analysis of KEGG differentially expressed miRNA target genes in adipose tissue;

Figure 3 is the verification result of differentially expressed miRNA qRT-PCR;

Figure 4 shows the results of qRT-PCR verification of differential expression of NC_040278.1_37602 and its target genes.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention, and should not be construed as a limitation of the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the claims and their equivalents. In the following examples, the experimental methods without specific conditions are usually detected according to conventional conditions or according to the conditions suggested by the manufacturer.

Example 1 Extraction of RNA from the collection agent of the sample

1.1 Collection of samples

In this experiment, Duolang sheep (D) and Xiaoweihan sheep (X) with differences in fat deposition were studied. The selected sheep are all adult females, and the individuals are in good physical condition, robust and disease-free, with a similar weight (about 50 kg), and each group has three biological replicates.

1.2 Extraction and quality inspection of sample RNA

Take an equal amount of adipose tissue from each sample for total RNA extraction, and ensure that the entire process is performed in a sterile environment. The steps are as follows:

(1) Grinding adipose tissue in a mortar with liquid nitrogen;

(2) put the ground tissue into the centrifuge tube with Trizol reagent and let stand;

(3) add chloroform and mix and let stand for 10 minutes at room temperature;

(4) Put it into a centrifuge, and centrifuge for 15 minutes at 4°C with a rotating speed of 12,000;

(5) Put the colorless liquid in the uppermost layer into the RNase free tube, use the same amount of isopropanol to precipitate the RNA, mix at room temperature and let stand for 10 minutes;

(6) Centrifuge again for 15 minutes at a temperature of 4°C and a rotating speed of 12000;

(7) Remove the supernatant, add ethanol (75%) to the precipitation and mix well, then centrifuge again under the above conditions;

(8) Remove the supernatant and continue to add ethanol to clean the RNA 2-3 times;

(9) discard ethanol, precipitation drying process 5-10 minutes;

(10) The final precipitate was treated with RNA enzyme-free water.

In order to ensure the purity and integrity of total RNA, it is subjected to quality inspection:

The requirement for library construction and sequencing is that the total amount of RNA is not less than 2ug. In this experiment, longRNA (mRNA, lncRNA, circRNA) and small RNA (miRNA) were constructed separately. The longRNA library was constructed using TruSeqTM Stranded Total RNAKit kit. The total amount of RNA was 2μg, the concentration was above 100ng/μL, and the OD260/280 range was 1.8-2.2. The small RNA library was constructed using the Illumina TruSeq Small RNA Kit.

Total RNA was extracted from 6 subcutaneous adipose tissues of Duolang sheep and Small-tailed Han sheep respectively, and the quality was tested. The results showed that the brightness of 28S band was significantly higher than that of 18S; the RNA concentration of each sample was higher than 200ng/μl; RIN The values are all greater than 8, OD260/280≥1.8, OD260/230≥1.0, indicating that the RNA is not polluted by impurities such as carbohydrates and proteins, and the integrity is good, which can be used in subsequent experiments.

Example 2 Sequencing and data analysis

Sequencing was performed using the Illumina NovaSeq 6000 sequencing platform. The quality control of longRNA-seq and smallRNA-seq data was performed with the software Fastp and Fastx_toolkit, respectively. The reference species used in the sequence alignment analysis was sheep (Ovis_aries), and the reference genome was obtained from the NCBI database (GCF_002742125.1). The reads matching the reference genome were compared with the miRNA precursor and mature sequences of sheep in the miRBase database, and the known miRNAs in each sample, including sequence information, length information, precursor sequence and position, were obtained. If there is no corresponding information in miRase for the species under study, the species with miRNA sequence information that is similar to the animal under study can be selected as the reference information for known miRNAs, and the miRNA sequence of the species with close relatives can be used to identify the known species of interest. miRNAs. A total of 868 miRNAs were obtained through statistics, of which the numbers of known and new miRNAs were 149 and 719, respectively.

Differential analysis of sample expression. There are obvious differences in the deposition of adipose tissue between Duolang sheep and Small-tailed Han sheep. By comparing the gene expression of the two breeds of sheep, the genes that are differentially expressed in the subcutaneous fat of the two breeds of sheep are screened out, so as to further study the significant difference in the fat deposition between the two breeds. molecular mechanism. The miRNAs and mRNAs (both containing known genes and newly identified genes) in the subcutaneous adipose tissue (D-PFvs X-PF) of Duolang sheep and Xiaoweihan sheep were screened strictly, and the differentially expressed genes that met the conditions were selected. About 95% of the screened miRNAs were filtered out, and there were 38 differentially expressed miRNAs, of which 10 were up-regulated and 28 were down-regulated. A total of 2439 mRNAs (1102 up-regulated and 1337 down-regulated) differed. The newly discovered miRNA (NC_040278.1_37602) was used as a candidate gene for subsequent verification.

In order to understand the functions of differentially expressed genes and gene products, GO annotation was used to annotate differentially expressed genes in three aspects, including biological processes, cellular components, and molecular functions. A total of 649 genes were annotated with GO Term in subcutaneous adipose tissue of Duolang sheep and Small-tailed Han sheep, and these genes were significantly enriched in 132 GO Term, including 101 biological processes (only GO Term with significant enrichment was selected). and Term closely related to fat development, lipid metabolism and related diseases), 14 cellular components and 17 molecular functions. As shown in Figure 1, the target genes were differentially expressed in terms of molecular functions, mainly enriched in heparin binding, C-8sterol isomerase activity, and glycerate kinase activity. And enzymatic activity regulation process such as glycosylphosphatidylinositol phospholipase D activity. In terms of cellular components, it was significantly enriched in related items such as cell surface, extracellular matrix, and plasma membrane part. In terms of biological processes, it is significantly enriched in the regulation of protein phosphorylation, the regulation of lipid catabolic process, the regulation of MAPK cascade, and the regulation of glycerol. Response to triglyceride, response to insulin (responseto insulin), etc. GO analysis showed that the differential miRNAs mainly regulated the activities of enzymes in the process of adipocyte differentiation and lipid metabolism in Duolang sheep and Small-tailed Han sheep, resulting in differences in fat deposition between the two breeds.

KEGG enrichment was used to analyze the function of target genes, and a total of 284 pathways were enriched. After setting Padjust<0.05, the pathways were screened, and a total of 43 significantly enriched pathways were obtained (Figure 2). The significantly enriched pathway is the PI3K-Akt signaling pathway, in addition to AMPK, steroid biosynthesis (Steroid biosynthesis), insulin (Insulin signaling pathway), starch and sucrose metabolism (Starch and sucrosemetabolism), phospholipase D ( It can be seen that differentially expressed miRNAs may regulate fat development by altering signal transduction.

Network analysis of targeting relationship between miRNA and mRNA. In order to observe the interaction between miRNA and mRNA more directly, we selected genes that were significantly enriched in GO and KEGG and were related to adipocyte differentiation, lipid metabolism and related diseases to draw a network map. Among them, oar-miR-493-3p, as the most connected gene in the network, targeted 15 mRNAs. The target genes of NC_040278.1_37602 gene are TBC1D19, PDE3B, CLIC4, PSD3, VAV3, TASOR2, SPATA6, PTGS2 and other genes.

Embodiment 3 Real-time fluorescent quantitative PCR detection verification

3.1 Reliability verification of sequencing results

In this experiment, the differentially expressed miRNAs were randomly selected between the two comparison groups (Duolang sheep and Small-tailed Han sheep) for PCR detection, each gene was replicated 3 times, and the expression trend of each miRNA was verified. The main methods are as follows:

reverse transcription

(1) Reverse transcription. The reverse transcription system using the kit was: total RNA 0.5 μg, 2×TS miRNA ReactionMix 5 μl, TransScrip miRNA RT Enzyme Mix 0.5 μl, Nuclease-free enzyme-free water was added to 10 μl. Setup program: 37°C for 60 minutes on an ABI 9700 PCR machine, and the reaction was terminated at 85°C for 5 seconds.

Table 2 PCR reaction system

Dispense the prepared PCR Mix into a 384-well plate, add 1 μl of the corresponding cDNA in turn, and make 3 replicate wells for each fat sample. After the film was scraped off, it was shaken and mixed, and centrifuged at 2000 rpm for 1 minute. After the end, take out the 384-well plate. The PCR program was: 94°C for 30 seconds; 94°C for 5 seconds, 60°C for 30 seconds, 45 cycles. After completion, the product specificity was tested. From 60 °C to 97 °C, the temperature needs to be raised slowly.

After the reaction, the data were derived, the relative expression of the samples was calculated by the 2- ^ΔΔCt method, and the relative expression was statistically analyzed by t-test. The data were shown as mean±standard deviation (Mean±SD).

PCR data analysis and results

Five differentially expressed miRNAs were randomly selected for qRT-PCR verification. The results showed that NC_040278.1_37602, NC_040253.1_5757, NC_040262.1_21536, NC_040278.1_37507 were up-regulated in the subcutaneous adipose tissue of Duolang sheep, and NC_040255.1_11631 was up-regulated in Dulang sheepskin subcutaneous adipose tissue. Down-regulated expression in lower adipose tissue (Figure 3). In the figure: * means P<0.05, ** means P<0.01, *** means P<0.001, the above results are consistent with the sequencing results, and it can be seen that the sequencing results are reliable.

3.2 Validation of candidate genes

In this experiment, 20 Duolang sheep and Small-tailed Han sheep were collected respectively, and the differentially expressed NC_040278.1_37602 and its target genes were detected by PCR between the two groups. Each gene was replicated three times to verify NC_040278.1_37602 and its target genes. For differential expression, please refer to 3.1 for experimental steps.

PCR data analysis and results, the relative expression of NC_040278.1_37602 and its target gene PTGS2 in each sample was calculated by the 2- ^△△Ct method. The genes differed significantly between the two groups. The results showed that the NC_040278.1_37602 gene was significantly down-regulated in the subcutaneous adipose tissue of Duolang sheep, and the PTGS2 gene was significantly up-regulated in the subcutaneous adipose tissue of Duolang sheep (see Figure 4 for details).

table 3

The genes closely related to fat deposition were screened by functional annotation and enrichment analysis, and the target relationship with differential miRNAs was predicted. This study found an unknown miRNA: NC_040278.1_37602, which functions by targeting the PTGS2 gene. Studies have shown that PTGS2 (prostaglandin-endoperoxidase synthase 2) in Angus and Chinese Simmental cattle adipose tissue This study found that NC_040278.1_37602 regulates the metabolism of subcutaneous fat and the differentiation of adipocytes in Duolang sheep and Small Tail Han sheep by targeting PTGS2 to regulate the fat deposition in Duolang sheep and Small Tail Han sheep. , and the results of real-time quantitative PCR experiments further verified the conclusion.

sequence listing

<110> Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences

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Claims (4)

1. The application of the reagent for detecting the expression level of the NC-040278.1-37602 sequence in the subcutaneous adipose tissues of sheep in preparing the detection reagent related to the subcutaneous fat content of the sheep is characterized in that the NC-040278.1-37602 sequence is shown in SEQ ID NO.1, and the sheep is small tailed han sheep or billow sheep.

2. The use of claim 1, wherein specific primers are used to amplify the NC 040278.1 37602 sequences.

3. The use of claim 2, wherein the primer sequence that amplifies the NC 040278.1 37602 sequence is SEQ ID No. 3.

4. The application of the kit for detecting the expression level of the NC-040278.1-37602 sequence in the subcutaneous adipose tissue of the sheep in preparing a detection reagent related to the subcutaneous fat content of the sheep is characterized in that the NC-040278.1-37602 sequence is shown in SEQ ID NO.1, and the sheep is small tailed han sheep or billow sheep.

Images