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CircRNA marker related to apoptosis of dairy cow mammary epithelial cells and application thereof

A technology of mammary epithelial cells and markers, applied in the field of circRNA markers, can solve the problems of decreased milk production and economic loss in the dairy industry, and achieve the effect of good application prospects.

Inactive Publication Date: 2021-04-09
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to research, under the condition of heat stress, the milk production of dairy cows decreases by 20% to 45%, which has caused huge economic losses to my country's dairy industry.

Method used

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  • CircRNA marker related to apoptosis of dairy cow mammary epithelial cells and application thereof
  • CircRNA marker related to apoptosis of dairy cow mammary epithelial cells and application thereof
  • CircRNA marker related to apoptosis of dairy cow mammary epithelial cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A method for identifying cow circular RNA, comprising the following steps:

[0043] Step 1: In a large dairy farm in Guangdong, healthy dairy cows from two groups of winter temperature (December 2018) and summer heat stress (August 2019) were selected as research objects; three dairy cows were selected in each of the two periods Breast tissue, tissue specimens were quickly frozen in liquid nitrogen for 30 seconds after being taken out, and then stored in a -80°C refrigerator, part of which was used for the extraction of tissue gDNA (whole genome DNA) and total RNA, and part of which was attached to dry ice and sent to the sequencing company for circRNA sequencing.

[0044] Step 2: Use the Shanghai Sangong Tissue gDNA Extraction Kit to extract the gDNA from the mammary gland tissue of cows. The specific operation is carried out according to the kit instructions. The extracted gDNA is stored in a -80°C refrigerator for later use; use the RNA extraction kit (Trizol method)...

Embodiment 2

[0057] The construction process of the cow circular RNA circbEZH2 overexpression vector is as follows:

[0058] The vector Pcd2.1-ciR empty vector (commercially available) is an engineering vector specially used for circular RNA expression, which is denoted as emptyvector, and contains a circular RNA expression framework such as Figure 5 As shown, after amplification of Escherichia coli DH5α, the vector DNA was extracted using the endotoxin plasmid mini-extraction kit. For specific steps, see the kit instruction manual.

[0059] Amplification process of the full-length sequence of CircbEZH2: use the full-length sequence of circbEZH2 as a template, use Primer5 software to design primers capable of full-length amplification, and the 5′ ends refer to KpnI and BamHI restriction sites and protective bases respectively, and the sequences are as follows Shown:

[0060] The sequence of the upstream primer (PF3): 5'-ggggtacctgaaatatgctatcttacagaataatcatgggccagact gg-3' (SEQ ID NO: 7)...

Embodiment 3

[0074] Cow mammary gland epithelial cells are MAC-T cell lines, cell transfection process:

[0075] Step 1: 1 day before transfection, use 5*10 4 Cells were seeded in a 12-well plate, and cultured until the cell density reached 80%;

[0076] Step 2: Add 1 μg of transfected plasmid per well (recombinant plasmid OE-circbEZH2 or emptyvector in Example 2) to 125 μL Opti-MEM medium and mix well, take 2 μL Lipofectamine 2000 and add 125 μL Opti-MEM medium and mix well , stand at room temperature for 5 minutes;

[0077] Step 3: Mix the two mixtures in Step 2 and let stand at room temperature for 20 minutes;

[0078] Step 4: Absorb the original cell culture medium in each well, and wash twice with Opti-MEM medium;

[0079] Step 5: Add 250 μL of the mixture in step 3 to each well of cells, and then make up to 1000 μL with Opti-MEM medium;

[0080] Step 6: Place at 37°C, 5% CO 2 The cells are cultured in an incubator, waiting for subsequent processing.

[0081] After 48 hours, the...

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Abstract

The invention discloses a circRNA marker related to apoptosis of dairy cow mammary epithelial cells and application thereof. The circRNA marker is circbEZH2, and the sequence of the circbEZH2 is shown as SEQ ID NO: 1. Whether the circbEZH2 forms a ring or not and the effect of an overexpression vector in the process of detecting the apoptosis resistance of the dairy cow mammary cells are verified, the overexpression vector plasmid of the circbEZH2 is transfected into the dairy cow mammary epithelial cells, the overexpression circbEZH2 can inhibit apoptosis of the dairy cow mammary epithelial cells, a new thought is provided for further analyzing the genetic mechanism of growth and development of the dairy cow mammary cells, deeper cognition is brought to the molecular mechanism of regulating and controlling the lactation characters of dairy cows through circular RNA, and the circRNA marker is applied to research of the molecular regulation and control mechanism of heat stress resistance of livestock and has good application prospects; and the circRNA marker has important significance in researching lactation regulation and control of the dairy cows and improving the dairy cow quality.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a circRNA marker related to the apoptosis of dairy cow mammary gland epithelial cells and its application. Background technique [0002] With the greenhouse effect and global warming, heat stress has become one of the most important problems in the production and breeding of dairy cows. At present, my country's dairy cows are mainly Holstein cows from the Netherlands, which are typical animals that are resistant to cold and heat. Underdeveloped sweat glands, poor heat dissipation, and a large amount of metabolic heat during lactation lead to heat intolerance of dairy cows to high temperature weather, and are prone to heat stress in summer, especially in southern my country. According to the research, under heat stress conditions, the milk production of dairy cows decreases by 20% to 45%, which has caused huge economic losses to my country's dairy industry. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/113C12N15/85
CPCC12Q1/6888C12N15/85C12Q2600/158C12Q2600/178C12N2800/107
Inventor 孙加节汪东阳张永亮习欠云陈婷罗君谊赵正江
Owner SOUTH CHINA AGRI UNIV
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