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Identification of isolation cloning and core region of promoters suitable for gene expression of skeletal muscles in pigs

A gene expression and promoter technology, applied in DNA/RNA fragments, recombinant DNA technology, animal husbandry, etc., can solve the problems of pork quality decline, muscle fat decline, muscle fiber thickening and fast muscle ratio, etc., to improve reliability and authenticity effect

Inactive Publication Date: 2011-02-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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.

Method used

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  • Identification of isolation cloning and core region of promoters suitable for gene expression of skeletal muscles in pigs
  • Identification of isolation cloning and core region of promoters suitable for gene expression of skeletal muscles in pigs
  • Identification of isolation cloning and core region of promoters suitable for gene expression of skeletal muscles in pigs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Verification of expression profiles of pig muscle-specific expression candidate genes (Genbank accession number: GU225952) in different tissues

[0045] 1. Main reagents: Taq DNA polymerase (product of Fermentas company), dNTP (Roche company, 4738284001), DL-2000 Marker (Guangzhou Dongsheng Biotechnology Co., Ltd.), AC column type quick agarose DNA recovery kit (Bio Teke company product, DP1701); Total RNA extraction reagent is Trizol Reagent (Invitrogen company product, Cat no: 15596-026); ReverTra Ace qPCR RT Kit (product of TOYOBO, Code no: FSQ-101) reverse transcription kit.

[0046] 2. Tissue samples: Heart, liver, spleen, lung, kidney, stomach, bone marrow, bladder, uterus, ovary, testis, epididymis, back fat, brain, small intestine, 20 tissue samples of lymph node, skin, longissimus dorsi, abdominal fat, and biceps femoris were placed in liquid nitrogen immediately after collection, and then stored at -80°C for the extraction of total RNA.

[0047]...

Embodiment 2

[0053] Example 2: Acquisition of the pig muscle-specific expression promoter Six1-P and the corresponding deletion fragment

[0054] 1. Main reagents:

[0055] 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 Co., Ltd. (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-2000 Marker (purchased from (from Guangzhou Dongsheng Biotechnology Co., Ltd.), AC Column Fast Agarose DNA Recovery Kit (purchased from Bio Teke Company, DP1701), carrier pMD18-T Vector (purchased from Bio-Technology (Dalian) Co., Ltd., Cat. No.: D101A) etc.

[0056] 2. Extraction of genomic DNA from pig longissimus dor...

Embodiment 3

[0090] Example 3: Construction of the transfection vector for the corresponding deletion fragment of the pig muscle-specific expression promoter

[0091] 1. Main reagents:

[0092] Endonucleases KpnI and XhoI (Fermentas, Cat.#ER0527 / Cat.#ER0691), Plasmid Extraction Kit OMEGA (OMEGA, Cat.D6950-01), T4 ligase (Fermentas, Cat.#EL0334), pGL3-Basic Vector (Promega, Cat. #E1751).

[0093] 2. Double enzyme digestion of the dual luciferase reporter vector pGL3-Basic

[0094] Digest the pGL3-Basic vector with KpnI and XhoI, double enzyme digestion system 20μl: 2μl 10×Tang TM Buffer, 2 μl KpnI, 2 μl XhoI, 4 μl pGL3-Basic vector, supplemented with sterilized ultrapure water to 20 μl. Digest overnight at 37°C. The digested product was recovered and purified with a purification kit (DP1701, purchased from Beijing Biotech Biotechnology Co., Ltd.). The recovered product was stored in a -20°C refrigerator.

[0095] 3. Double enzyme digestion of the TA cloning vector containing the corr...

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Abstract

The invention belongs to the field of animal gene engineering. Seven promoters Six1-P, P1, P2, P3, P4, P5 and P6 with different lengths in the upstream of a specific expression gene of skeletal muscles in pigs are cloned from a pig genome. Nucleotide sequences of the promoters are shown in SEQ ID NO:1; 2; 3; 4; 5; 6; and 7. The lengths of the nucleotide sequences are respectively 1,692, 180, 530, 844, 1,143, 1,135 and 1,494bp. The activity analysis on promoter fragments with different lengths of a pig Six1 gene in three eukaryotic cells shows that the promoters of the pig Six1 gene are promoters which express the muscle specificity; except for the P1, other promoters P2, P3, P4, P5 and P6 all have the capacity of promoting gene expression; and in the promoters P2, P3, P4, P5 and P6, except for the promoter P2, other promoters all show the muscle specificity. The invention discloses the seven promoters, a method for preparing corresponding expression vectors of the seven promoters and identification of the promoter activity of the pig Six1 gene by using a dual-luciferase reporting system.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to the isolation and cloning of a promoter suitable for pig skeletal muscle gene expression and the identification of its core region. 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. Although gene expression is a multi-level regulatory system in higher organisms, transcription, as the...

Claims

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Application Information

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IPC IPC(8): C12N15/113A01K67/027
Inventor 熊远著吴望军雷明刚左波任竹青徐德全
Owner HUAZHONG AGRI UNIV
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