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A kind of circRNA, product and application thereof

A product and sample technology, applied in the field of agricultural genetic engineering, can solve problems such as fat buttocks, and achieve the effect of improving meatiness and meat performance.

Active Publication Date: 2022-04-15
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are few studies on sheep subcutaneous fat. In order to further discover new circRNAs related to sheep subcutaneous fat, the inventors selected representative Duolang sheep with large differences in fat deposition based on high-throughput sequencing technology. (D) and the small-tailed Han sheep (X) as the research object, the former belongs to the meat and fat dual-purpose sheep, with a large fat buttocks, and the latter belongs to the short and thin tail sheep, with less subcutaneous fat. Key candidate genes for differentiation to explore molecular mechanisms related to fat deposition

Method used

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  • A kind of circRNA, product and application thereof
  • A kind of circRNA, product and application thereof
  • A kind of circRNA, product and application thereof

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Experimental program
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Effect test

Embodiment 1

[0040] The collecting agent RNA of embodiment 1 sample is extracted

[0041] 1.1 Collection of samples

[0042] In this experiment, Duolang sheep (D) and Small Tail Han sheep (X) with different fat deposits were studied. The selected sheep are all adult females, and the individuals are in good body condition, strong and disease-free, with similar body weight (about 50kg), and each group has three biological replicates.

[0043] 1.2 Extraction and quality detection of sample RNA

[0044] Take an equal amount of adipose tissue from each sample to extract total RNA, and ensure that the whole process is carried out in a sterile environment. The steps are as follows:

[0045] (1) Grinding adipose tissue in a mortar with liquid nitrogen;

[0046] (2) Put the ground tissue into a centrifuge tube added with Trizol reagent and let it stand;

[0047] (3) Add chloroform and mix well and let stand at room temperature for 10 minutes;

[0048] (4) Put it into a centrifuge, and centrifu...

Embodiment 2

[0058] Example 2 Sequencing and Data Analysis

[0059] Sequenced using the Illumina NovaSeq 6000 sequencing platform. circRNA is a closed ring, which is formed by the reverse connection of the exon splicing acceptor and the donor site located upstream and downstream respectively. more stable. Linear RNA fragments can be aligned by linear alignment software, but Reads at the trans-splicing position of circular RNA cannot be directly aligned to the genome. Based on Back splicejunction (BSJ) reads, use related software (CIRI2+find_circ take union) to predict circRNA, and compare with database circBase (animal).

[0060] Classification is based on the start and end points of circRNAs on the genome. The first type of termination point and start point are located in the exon region of the gene as exon circRNA. The second type of starting point or ending point is located in the intron region of the gene as intron circRNA. When one end of the third type of starting point or endin...

Embodiment 3

[0066] Embodiment 3 real-time fluorescent quantitative PCR detection verification

[0067] In this experiment, 20 Duolang sheep and Small-tailed Han sheep were collected, and the differentially expressed circRNA was detected by PCR between the two groups. Each gene was replicated three times to verify the differential expression of the NC_040256.1_12467238_12540333 gene.

[0068] The main method is as follows:

[0069] Use circRNA cDNA Synthesis Kit (GenePool, Cat#GPQ1805) for reverse transcription, the reaction system is: 2×FastSYBR Mixture, 10 μl; downstream primer (10uM) and upstream primer (10uM) each 0.4 μl; template, 2 μl; sterilized distilled water to 20 μl. See the table for primer information.

[0070] Table 1 PCR reaction results

[0071]

[0072] To export the data after the reaction, use 2 -ΔΔCt The relative expression of the samples was calculated by the method, and the t-test was used to analyze the relative expression. The data were shown as mean ± standa...

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Abstract

The present invention relates to a circRNA, a product and its application, in particular to a circRNA for detecting sheep subcutaneous fat, a product and its application. Existing studies have shown that circRNA may be involved in the regulation of lipid metabolism and can provide potential targets for lipid metabolism-related diseases. In order to discover new circRNAs related to sheep subcutaneous fat, the inventors selected two representative sheep with large differences in fat deposition as research objects based on high-throughput sequencing technology, and screened and identified candidate genes related to lipid metabolism , and verified by molecular experiments, this application lays the foundation for sheep auxiliary breeding in animal husbandry and the like.

Description

technical field [0001] The invention relates to the technical field of agricultural genetic engineering, in particular to a circRNA for detecting sheep subcutaneous fat, a product and an application thereof. Background technique [0002] circRNA is a kind of closed circular RNA molecule first discovered in plant virus and influenza virus, which is produced by special shearing. circRNA does not have a 5' cap and 3' polyadenylation structure. Compared with linear RNA, circRNA is more stable because ribonuclease cannot degrade circRNA. Endogenous competing circRNAs can also affect miRNA activity, thereby regulating gene expression. It is speculated that circRNAs may regulate adipogenic differentiation through the above principles. The study found that 31 circRNAs were significantly increased during the adipose differentiation process in the in vitro differentiation system of preadipocytes. Correlation analysis showed that the expression of these circRNA molecules may be relate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/113C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/158C12Q2600/178
Inventor 苗向阳刘天义冯卉解领丽李嫒
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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