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Separation, cloning and identification of promoters of porcine citric dehydrogenase genes IDH3beta

A technology of isocitrate dehydrogenase and promoter, applied in the field of pig breeding and molecular biology

Inactive Publication Date: 2012-07-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, the cloning of the porcine IDH3β promoter, the analysis of the promoter activity and the mutation research of the promoter region are still blank at home and abroad.

Method used

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  • Separation, cloning and identification of promoters of porcine citric dehydrogenase genes IDH3beta
  • Separation, cloning and identification of promoters of porcine citric dehydrogenase genes IDH3beta
  • Separation, cloning and identification of promoters of porcine citric dehydrogenase genes IDH3beta

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Preparation of the upstream 5' flanking sequence of porcine citrate dehydrogenase gene IDH3β gene

[0074] The TAIL-PCR method (Liu et al., 1995) was used to isolate the promoter sequence of porcine citrate dehydrogenase gene IDH3β. This method required two rounds of PCR amplification for each step. The PCR reaction conditions are shown in Table 1.

[0075] Table 1. Three rounds of amplification conditions for TAIL-PCR

[0076]

[0077]

[0078] The reaction system is as follows: (1) First round: 100ng porcine genomic DNA, 0.2μM gene-specific primer 1 (SE), 2μM random primer R (R1, R2, R3 and R4), 2.5μL 2mM dNTP, 1U Taq DNA polymerase , 2.5 μL of 10×Taq DNA polymerase buffer, added to a total volume of 25 μL; (2) the second round. Dilute the first-round PCR product 50 times, take 1 μL as template, 0.2 μM SE gene-specific primer 2 (SF), 2 μM random primer R (R1, R2, R3 and R4), 2.5 μL 2mM dNTP, 1U Taq DNA polymerase , 2.5 μL 10×Taq DNA polymerase, adde...

Embodiment 2

[0095] Example 2: Detection method for the mutation of the upstream 5' flanking sequence of porcine citrate dehydrogenase gene IDH3β

[0096] Genomic DNA was extracted from 2 foreign blood-related pig breeds "Large White Pig" and 2 Chinese native pig breeds "Meishan Pig", and the 2447bp 5' flanking sequence of porcine citrate dehydrogenase gene IDH3β obtained according to Example 1 was used as the test material , design the following primers, the primer numbers and nucleotide sequences are as follows:

[0097] Forward primer 31: TGGCTCTGTCCAAGGGTT;

[0098] Reverse primer 32: AACTGGGATTTGTACTGC.

[0099] Use primers 31 and 32 to carry out PCR amplification in the genomic DNA of the above two large white pigs and two Meishan pigs. The PCR reaction system is 25 μl, in which the template DNA is 50 ng, the concentration of dNTPs is 200 μmol / L, and the concentration of each primer is 0.3 μmol / L, 1U Taq DNA polymerase (Biostar International, Canada), add deionized water to a tota...

Embodiment 3

[0101] Example 3: Construction of expression vector containing porcine citrate dehydrogenase gene IDH3β upstream 5' flanking sequence

[0102] 1. Primer Design

[0103] According to the characteristics of the multiple cloning site of the pGL3-Basic vector (purchased from Promega, USA) and the promoter sequence of the porcine ACL gene, primers were designed using the deletion method, and there were XhoI and MluI restriction sites at the 5' ends of the upstream and downstream primers, respectively. (The sequence of the restriction site is underlined, as shown in Table 3), and three protective bases are respectively added upstream of the recognition sequence of the restriction site to ensure that the restriction site is not lost. The large white pig genomic DNA was used as a template for PCR reaction, and the annealing temperature of PCR amplification was 58°C. The primer numbers and their nucleotide sequences are as follows:

[0104] Forward primer 11: GCCACGCGTGAGAGAGACGCTTTC...

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Abstract

The invention belongs to animal biology technology and breeding field of pigs, and particularly relates to cloning, activity analysis and promoter region mutation research of promoters of porcine isocitrate dehydrogenase genes IDH3beta. The method comprises the following steps of: extracting genomes DNA from porcine blood, designing primers, obtaining genome base sequences of upstream 5' flank 2447bp of porcine IDH3beta genes in two-time amplification mode by adopting TAIL-PCR technology, and performing sequence comparison and analysis and genetic variation detection; and amplifying the upstream 5' flank region of the IDH3beta genes by adopting 5'-end deletion strategy, constructing luciferase report gene vectors by using the obtained DNA sequences, measuring the activity of the promoters, and identifying the promoters of the IDH3beta genes. The nucleotide sequences of the promoters of the cloned porcine isocitrate dehydrogenase genes IDH3beta are expressed as sequence tables SEQ ID No: 1 and SEQ ID No: 4. The invention provides gene resources for porcine genetic improvement application.

Description

technical field [0001] The invention belongs to the technical field of pig breeding and molecular biology, and in particular relates to the isolation, cloning and identification of a porcine citrate dehydrogenase gene IDH3β promoter. Background technique [0002] China is the world's largest pig raising and pork consumption country. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), in 2007 China had 446.4 million pigs on hand, 620.8 million pigs on the market, and a total pork output of 47.2 million tons, accounting for 47.43%, 47.28% and 45.75% of the world respectively, ranking first in the world. At present, most of the pig breeds raised in large-scale pig farms in my country are foreign lean meat breeds, such as Duroc pigs, Large White pigs, and Landrace pigs. The urgent need; but at the same time in the pursuit of high lean meat rate also led to a decline in pork quality. Intramuscular fat (IMF) content is considered to be ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12Q1/68
Inventor 任竹青熊远著蒋思文邓昌彦
Owner HUAZHONG AGRI UNIV
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