Application of dairy cattle bta-miRNA29d-3p to dairy cattle mammary epithelial cell lipid accumulation regulation process

A technique for mammary epithelial cells and cows, which is used in DNA/RNA fragmentation, recombinant DNA technology, determination/inspection of microorganisms, etc.

Active Publication Date: 2021-08-06
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] This description overcomes the deficiencies in the prior art and provides an application of cow bta-miRNA29d-3p in the regulation of l...

Method used

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  • Application of dairy cattle bta-miRNA29d-3p to dairy cattle mammary epithelial cell lipid accumulation regulation process
  • Application of dairy cattle bta-miRNA29d-3p to dairy cattle mammary epithelial cell lipid accumulation regulation process
  • Application of dairy cattle bta-miRNA29d-3p to dairy cattle mammary epithelial cell lipid accumulation regulation process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Detection of the expression level of bovine miRNA gene bta-miRNA29d-3p in bovine mammary gland epithelial cells.

[0044] The following chemically synthesized miRNAs and their controls are provided by Guangzhou Ruibo Biotechnology Co., Ltd.:

[0045] (1) bta-miRNA29d-3p mimics (miR-29d), the sequence is:

[0046] 5'-UAGCACCAUUUGAAAUCGAUUA-3' (SEQ ID NO: 3)

[0047] 3'-AUCGUGGUAAACUUUAGCUAAU-5' (SEQ ID NO: 4)

[0048] (2) The negative control (ConmiR) of miRNA mimics, the sequence is: 5'-UUCUCCGAACGUGUCACGU-3'

[0049] (sequence 6);

[0050] (3) The sequence of bta-miRNA29d-3p inhibitor (anti-miR-29d) is:

[0051] 5'-UAAUCGAUUUCAAAUGGUGCUA-3'

[0052] (sequence 5);

[0053] (4) The negative control (ConInh) of miRNA inhibitor, the sequence is: 5'-CUCCGAACGAACGUGUCACGU-3' (SEQ ID NO: 7);

[0054] On this basis, LipofectamineTM RNAiMAX transfection reagent was used to transiently transfect miR-29d, ConmiR, anti-miR-29d, and Con Inh into bovine mammary epithe...

Embodiment 2b

[0091] Example 2 Effect of bta-miRNA29d-3p on the expression of lipid metabolism genes and triglyceride content in dairy cow mammary gland epithelial cells

[0092] 1. Detection of bta-miRNA29d-3p on the mRNA of lipid metabolism genes in cow mammary gland epithelial cells.

[0093] The operations of RNA extraction, reverse transcription, and fluorescence quantification were the same as in Example 1. The primers used are listed in the table below:

[0094]

[0095]

[0096] Such as Figure 2A , Figure 2B , Figure 2C The results showed that after overexpression of bta-miRNA29d-3p, the expression of transcription factor SREBF1 was significantly down-regulated, and the expression of LXRA was significantly up-regulated; the expression of fatty acid synthesis and elongation genes ACACA, FASN and ELOVL4 was significantly down-regulated, and the expression of triglyceride synthesis The expression of DGAT1 and GPAM was significantly down-regulated; the opposite result was o...

Embodiment 3b

[0101] Example 3 Verification of direct action of bta-miRNA29d-3p and bovine ELOVL43'UTR target site

[0102] 1. Prediction of bta-miRNA29d-3p-target genes

[0103] Using TargetScan7.1 (http: / / www.targetscan.org / vert_71 / ) to predict the downstream-target genes of bta-miRNA29d-3p, the results show that the gene related to fatty acid metabolism in the target genes is ELOVL4, and the results show that bta-miRNA29d -3p is relatively conserved in humans (Human) and chimpanzee (Chimpanzee) (such as Figure 4A shown).

[0104] 2. Construction of psiCHECK-2-ELOVL43'UTR recombinant plasmid

[0105] The bovine ELOVL43'UTR fragment (855bp) (sequence 33) was amplified by PCR technology, cloned into the XhoI / NotI restriction site of the dual luciferase reporter gene vector psiCHECK-2vector after digestion, and constructed to contain wild-type bovine ELOVL43' Dual-luciferase reporter gene vector for UTR sequences. (SEQ ID NO: 34) At the same time, a dual luciferase expression ...

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Abstract

The invention belongs to the field of dairy cattle breeding genetic engineering, and particularly relates to a dairy cattle miRNA gene bta-miRNA29d-3p, and functional verification of the gene shows that after the gene is over-expressed, a key gene of a fatty acid synthesis and metabolism pathway and a key gene of a triglyceride synthesis and metabolism pathway can be inhibited, and meanwhile, the content of triglyceride in cells is reduced; after the gene is interfered, the key gene of the fatty acid synthesis and metabolism pathway and the key gene of the triglyceride synthesis and metabolism pathway can be up-regulated, the content of the triglyceride in the cells is obviously up-regulated, and the bta-miRNA29d-3p directly acts on a fatty acid elongase ELOVL4 gene. Based on such technical effect, a foundation can be laid for regulation of the content of milk fat in the milk of dairy cattle and breeding of high-milk fat varieties.

Description

technical field [0001] This specification relates to the field of genetic engineering of dairy cow breeding, and more specifically relates to the application of a dairy cow bta-miRNA29d-3p in the process of regulating lipid accumulation in cow mammary gland epithelial cells. Background technique [0002] Milk not only provides energy, but also contains high-quality protein, vitamins and calcium, and is an important source of nutrients in the national diet. Milk has a high total fat content and is rich in fatty acids, among which unsaturated fatty acids have a good preventive effect on human cardiovascular diseases and hyperlipidemia. Studies have shown that milk quality is also closely related to the composition and content of fatty acids. Therefore, improving the lipid composition and lipid content in milk is particularly important to improve milk quality, and increasing the milk fat content in milk has become one of the important goals of current dairy breeding work. [...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/113
CPCC12Q1/6888C12Q2600/158C12Q2600/178C12Q2600/124
Inventor 姚大为杨春蕾张效生马毅李玉鹏
Owner 天津市农业科学院
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