Swine gene expression muscle creatine kinase (MCK)-diacylglycerol acyltrabsferase 1(DGAT1) carrier and preparation method thereof

A technology of MCK-DGAT1 and expression vectors, which is applied in the field of preparation of MCK-DGAT1 vectors, can solve the problems of pork quality decline, muscle fat decline, muscle fiber thickening, etc., to improve reliability and authenticity, increase triglyceride and The effect of intramuscular fat content

Inactive Publication Date: 2013-06-05
HUAZHONG AGRI UNIV
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  • Abstract
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Problems solved by technology

But at the same time, with the intensity selection of high growth rate and high lean meat rate, the quality of pork is seriously reduced, especially the muscle fat is greatly reduced, the muscle fiber is thickened (the proportion of fast muscle is increased), and a large number of inferior pork emerge. (Bee G, Guex G, Herzog W; Pettigrew J E, Esnaola MA, 2001)

Method used

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  • Swine gene expression muscle creatine kinase (MCK)-diacylglycerol acyltrabsferase 1(DGAT1) carrier and preparation method thereof
  • Swine gene expression muscle creatine kinase (MCK)-diacylglycerol acyltrabsferase 1(DGAT1) carrier and preparation method thereof
  • Swine gene expression muscle creatine kinase (MCK)-diacylglycerol acyltrabsferase 1(DGAT1) carrier and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The promoter of pig muscle specific expression gene MCK (Genbank accession number: AC139878) and pig DGAT1 gene (Genbank accession number: NM-214051.1) constitute an expression vector.

[0040] 1. Main reagents: Taq DNA polymerase (product of Fermentas Company), dNTP (Roche Company, 4738284001), DL-2000Marker (Guangzhou Dongsheng Biotechnology Co., Ltd.), TIANgen Mini Agarose Gel DNA Recovery Kit (Tiangen Company product, DP208-02), restriction enzymes SnaB I, Sal I, Nde I, Ase I, Not I, PspOM I, Swa I (NEB products, R0130S, R0138S, R0111S, R0526S, R0653S, R0646S), T4DNA ligase (product of NEB Company, M0202S), TIANprep Mini Plasmid Small Extraction Kit, product of Tiangen Company, DP103-02), vector pMD18-T Vector (purchased from Treasure Bioengineering (Dalian) Co., Ltd., item number: D101A), etc. .

[0041] 2. Tissue sample: porcine muscle tissue DNA

[0042] 3. Build the carrier:

[0043] With the pig MCK gene cDNA sequence, its Genbank accession number is AC13987...

Embodiment 2

[0054] Extraction of total RNA from tissue of transgenic mice, synthesis of cDNA, analysis of tissue expression and detection by fluorescent quantitative PCR.

[0055] 1. Main reagents:

[0056] The DNA extraction kit used in the experiment (purchased from Bio Teke Company, article number: DP1901); TransEco FastPfu DNA Polymerase (purchased from Beijing Quanshijin Biotechnology Co., Ltd., article number: AP231); 2 × GC Buffer (purchased from Bao Biological Engineering ( Dalian) Co., Ltd., product number: DRR20GCI), LA Taq polymerase (purchased from Bao Biological Engineering (Dalian) Co., Ltd., product number: DRR20BG), dNTP (purchased from Roche Company, product number: 47382001), DL-2000Marker (purchased from Guangzhou Dongsheng Biotechnology Co., Ltd.), TIANgel Mini Agarose Gel DNA Recovery Kit (product of Tiangen Company, DP208-02), etc.

[0057] 2. Extraction of total RNA from transgenic mouse tissue and synthesis of cDNA:

[0058] The total RNA of each tissue was extra...

Embodiment 3

[0067] Detection of protein level in transgenic mice and determination of triglyceride content:

[0068] 1. Main reagents:

[0069] RIPA lysate (purchased from Biyuntian Company, product number: P0013D), PMSF (purchased from Biyuntian Company, product number: ST506), DGAT1 primary antibody (purchased from abcam company, product number: ab59034), HRP rabbit anti-goat secondary antibody (purchased From antgene company, article number: ANT013), pre-stained protein marker (purchased from Fermentas company, article number: SM0441), 2*Loading Buffer (purchased from Tiangen company, article number), OCT embedding agent (purchased from Sakura Finetek company, article number: 4583) etc.

[0070] 2. Western Blot

[0071] (1) Collection of protein samples (Protein sample preparation)

[0072] Biyuntian RIPA lysate can be used to lyse tissue samples. After the protein samples were collected, in order to ensure that the loading amount of each protein sample was consistent, the protein ...

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Abstract

The invention discloses a swine gene expression muscle creatine kinase (MCK)-diacylglycerol acyltrabsferase 1(DGAT1) carrier and a preparation method thereof. The preparation method includes a first step of obtaining a complementary deoxyribonucleic acid (c DNA) overall length sequence of a swine DGAT1 gene according to the DGAT1 gene to compound the gene, a second step of designing two pairs of two-end distinguished sequences of two ends of a specific primer augmentation swine MCK gene promoter, enabling the two-end distinguished sequences to be connected on a p enhanced green fluorescent protein (p EGFP)-N1 carrier, obtaining the complete promoter of the swine MCK gene according to gene capture technology, wherein the overall length of a promoter sequence is 7121bp, and a third step of using the swine DGAT1 gene to replace a green fluorescent protein (GFP) gene on a p EGFP-N1-MCK carrier, and completing the structure of the MCK-DGAT1 expression carrier. The experimental result indicates that the expression carrier structured by the swine DGAT1 gene and the muscle specific expression gene MCK promoter, the contents of triglyceride and aliphatic acid in animal individual muscles are remarkably increased, and reliability and authenticity of the experiment are improved. The verified expression carrier is directly applied to swine transgenic engineering and molecular breeding. Through transgenosis, new species of high intramuscular fat can be obtained, quality of pork is improved, and the time for conventional breeding cultivating high intramuscular adipose is shortened.

Description

technical field [0001] The invention belongs to the technical field of animal gene engineering, and in particular relates to an MCK-DGAT1 carrier for pig gene expression, and also relates to a preparation method for the MCK-DGAT1 carrier for pig gene expression. Background technique [0002] Gene expression refers to the process from DNA to protein, and the regulation of this process is called gene expression regulation. The regulation of gene expression is a multi-level complex process, which is controlled by different regulatory factors. It is achieved through multi-stage level regulation, that is, pre-transcriptional, transcriptional, post-transcriptional, translational, and post-translational levels of regulation. The growth and development of higher organisms is realized by the orderly expression and synergy of different genes in time and space. Fine control of levels. Promoter refers to a DNA sequence that enables gene transcription. If the structural gene is under ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66
Inventor 熊远著黎婷雷明刚左波徐德全
Owner HUAZHONG AGRI UNIV
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