CircRNA expression profile related to generation and development of sheep hair follicles as well as construction method and application of circRNA expression profile

A construction method and expression profiling technology, applied in the field of circRNA expression profiling and construction related to hair follicle development, can solve the problems of lack of research on the regulation of circRNA development

Pending Publication Date: 2022-01-11
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

circRNA has been studied in a variety of tissues and cells, however, research on the role of circRNA in the regulation of hair follicle development in cashmere goat embryos is still very scarce

Method used

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  • CircRNA expression profile related to generation and development of sheep hair follicles as well as construction method and application of circRNA expression profile
  • CircRNA expression profile related to generation and development of sheep hair follicles as well as construction method and application of circRNA expression profile
  • CircRNA expression profile related to generation and development of sheep hair follicles as well as construction method and application of circRNA expression profile

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Experimental program
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Effect test

Embodiment 1

[0040] A method for constructing a hair follicle development-related circRNA, comprising the following steps:

[0041] (1) Prepare test animals and samples

[0042] Three-year-old ewes were selected from Jinlai Animal Husbandry in Inner Mongolia for simultaneous estrus treatment, and the mating time was recorded. After taking three sheep skin samples at 45 days, 55 days, 65 days and 75 days of the fetus, they were immediately treated with DPEC water and placed in liquid nitrogen. Then store in a -80°C refrigerator for RNA-seq and RT-qPCR experiments.

[0043] (2) RNA library construction and sequencing

[0044] Total RNA was isolated and purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA quantity and purity of each sample were quantified using NanoDrop ND-1000 (NanoDrop, Willington, DE, USA). RNA integrity was assessed with an Agilent 2100. Use approximately 5 µg of total RNA to deplete ribosomal RNA TM The rRNA...

Embodiment 2

[0069] Example 2 CircRNA3236 targets binding chi-miR-27b-3p and chi-miR-16b-3p

[0070] circRNA3236 was down-regulated in d75vsd45, and Targetscan and miRanda software predicted that circRNA3236 targets chi-miR-27b-3p and chi-miR-16b-3p, which have two binding sites respectively. Therefore, two mutant vectors were constructed to verify specific binding sites ( Figure 7 A and Figure 7 B). The results show that in this experiment, there is a binding effect between the two. After mu1 mutation, chi-miR-27b-3p failed to down-regulate the expression of luciferase in circRNA3236-mut1 (p>0.05), indicating that the mutation was successful. Mu1 is the binding site between chi-miR-27b-3p and circRNA3236. After mutation, chi-miR-16b-3p failed to down-regulate the expression of luciferase in circRNA236-mut4 (p>0.05), indicating that the mutation was successful. Mu4 is the binding site of chi-miR-16b-3p and circRNA3236 ( Figure 7 C and Figure 7 D).

[0071] Specific steps for dua...

Embodiment 3

[0073] In order to further explore the functional mechanism of circRNAs in the development of hair follicles in cashmere goats, the miRNAs targeted by circRNAs differentially expressed in d75vsd45 and the target genes targeted by miRNAs were predicted to construct a ceRNA network. Among them, there are 16 circRNAs targeting 6 miRNAs and 6 miRNAs targeting 23 target genes in the up-regulation-down-up-regulation mode, and 14 circRNAs targeting 16 miRNAs and 16 miRNAs targeting 26 target genes. Constructed circRNA9127-chi-miR-1-WNT3, circRNA2046-chi-miR-93-3p-FLRT1,

[0074] circRNA2962-chi-miR-20b-SMAD6, circRNA3236-chi-miR-27b-3p-SFRP1, circRNA3236-chi-miR-16b-3p-SLITRK3, circRNA1476-chi-miR-10b-3p-WNT2 and other signaling pathways. Among them, SFRP1, WNT3, SMAD6, and other genes all come from important pathways related to hair follicle development in previous studies, including WNT, TGF-β, TNF and other signaling pathways. Therefore, it is further speculated that circRNA, as...

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Abstract

The invention provides a circRNA (Ribonucleic Acid) expression profile related to generation and development of sheep hair follicles as well as a construction method and application of the circRNA expression profile. The construction method comprises the following steps: (1) RNA library construction and sequencing: constructing an RNA library for a sample of body side skin of a sheep in a fetal period, and sequencing; (2) identifying a transcript: predicting circRNA according to a circRNA identification standard; (3) differential expression analysis: carrying out differential expression analysis on the circRNA identified in the step (2); (4) GO enrichment and KEGG pathway analysis: carrying out GO enrichment and KEGG pathway analysis on the differentially expressed circRNA related host genes in the step (3); and (5) construction of a ceRNA regulation and control network: predicting the circRNA targeted miRNA, predicting a target gene of the targeted miRNA, and preliminarily constructing a circRNA-miRNA-mRNA regulation and control network. An important basis is provided for exploring a molecular mechanism of circRNA as ceRNA in generation and development of hair follicles in the future.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a circRNA expression profile related to hair follicle development and its construction method and application. Background technique [0002] There are two types of hair follicles in the skin of cashmere goats, namely primary hair follicles and secondary hair follicles. Primary hair follicles produce coarse hairs, and secondary hair follicles produce downy hairs. The skin and hair follicle structure characteristics of cashmere goats are not only the important content of their biological characteristics, but also have a direct and important impact on the yield and quality of cashmere goats. The skin hair follicle traits of cashmere goats have a direct and important impact on the yield and quality of hair follicles. The ultimate goal of research in the field of hair follicle growth and development in cashmere goats is to reveal the mechanism of hair growth and find important genes rela...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C12Q1/6888C12N15/10C12N15/113
CPCC12Q1/6806C12Q1/6869C12Q1/6888C12N15/1072C12N15/113C12N2310/532C12Q2535/122C12Q2525/307
Inventor 张燕军尚方正马荣
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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