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Regulatory Sequence of Porcine Liver Carboxylesterase Gene

A pig liver carboxylesterase, pig genome technology, applied in genetic engineering, plant gene improvement, enzymes and other directions, can solve problems such as restricting the research and application of PLE gene regulation mechanism, disordered sequence, and inability to provide sufficient basis for research.

Inactive Publication Date: 2019-04-05
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the research on PLE has been relatively sufficient, there is still a big gap in the data on the PLE gene sequence, especially the research on the regulatory mechanism. The PLE gene sequence provided in the authoritative database is incomplete or in disordered order, which cannot provide information for further research. Sufficient evidence seriously restricts the follow-up research and application of PLE gene regulation mechanism

Method used

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  • Regulatory Sequence of Porcine Liver Carboxylesterase Gene
  • Regulatory Sequence of Porcine Liver Carboxylesterase Gene
  • Regulatory Sequence of Porcine Liver Carboxylesterase Gene

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1: Primer Verification

[0048] Using the genome of Tongcheng pig (a common local pig breed in Hubei) as a template (the genomic DNA sample of Tongcheng pig was donated by Professor Liu Bang, School of Animal Science and Technology, Huazhong Agricultural University), and using the primer pair (ID1F, ID1R) in the above table 1 For PCR amplification, screen suitable primers according to the presence and size of the product, and the PCR reaction system is shown in Table 3. Amplification conditions: pre-denaturation: 94°C for 5min, denaturation: 94°C for 30s, annealing: 62°C for 1min, extension: 72°C for 4min, number of cycles: 30cycles, extension: 72°C for 10min. After PCR amplification was completed, the products were detected by 0.8% agarose gel electrophoresis. From the agarose gel electrophoresis pattern, it can be seen that the size of the amplified band is similar to the expected target product and the band is brighter, indicating that the pair of primers is...

Embodiment 2

[0051] Embodiment 2: Cloning, screening PLE1 gene

[0052] Currently, there are two methods for screening target genes in BAC library: hybridization method and PCR screening method. PCR screening method is a routine method used in the laboratory and the operation procedure is relatively flexible. This embodiment adopts a three-step PCR screening method. The selected primers were used to screen the PLE gene in the established Rongchang pig BAC library (Liu, 2010).

[0053] The specific operation steps are as follows:

[0054] (1) Take out the BAC library stored in the -80°C refrigerator in advance and put it in the 4°C refrigerator to thaw, and inoculate all the BAC clones on a 384-well culture plate into an LB solid agar culture plate (additional 12.5 μg / mL chloramphenicol), cultivate overnight in a 37°C incubator, and observe the growth of colonies. Then add 5 mL of LB liquid medium on the LB agar culture plate (because the LB on the culture plate is a solid medium, adding...

Embodiment 3

[0061] Example 3: Plasmid extraction and identification of positive clones

[0062] Name the positive clone obtained by screening as BAC-70, transfer the BAC-70 positive clone bacteria solution to LB liquid medium (with 12.5 μg / mL chloramphenicol added), place it on a shaker at 37°C and 200 rpm, and shake the bacteria overnight (16h) Expand the culture, and use the BAC / PACDNA Isolation Kit (purchased from OMEGA, USA) to extract the plasmid according to the instructions. Detected with a NANODROP 2000C ultra-micro spectrophotometer (Thermo Scientific), the A260 / A280 is 1.96, and the A260 / A230 is 2.1. Take out 5 μL of plasmid samples and carry out electrophoresis identification on 0.8% agarose gel, set the voltage at 50V, electrophoresis at 4°C for 3 hours, the sample bands are complete, there is no impurity contamination and obvious degradation phenomenon, which meets the requirements of PacBio third-generation sequencing for samples. Then send it to Beijing Biomike Biotechnolo...

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Abstract

The invention relates to a regulatory sequence of a pig liver carboxylesterase gene. Analyzing incomplete PLE gene sequences on NCBI, designing primers, screening from a Rongchang swine BAC library, thus obtaining PLE gene positive clone, carrying out sequencing by adopting a PacBio three-generation sequencing method, carrying out splicing to obtain a complete sequence, and carrying out bioinformatics analysis, thus obtaining the regulatory sequence (-22,655bp to -1bp) of the PLE1 gene as shown in SEQ ID NO: 1. The Blast analysis shows that two sections of reverse complementary sequences of 720bps (the two sections of reverse complementary sequences are located at -5132 / -4413bp and -802 / -82bp of the regulatory sequence of the PLE1 gene) exist in the regulatory sequence of the PLE1 gene, the two sections of sequences form a Loop cyclic special structure, and the test proves that the special structure can up-regulate the transcription and expression of the PLE1 gene, and can be applied to the aspects of PLE1 medicine hydrolysis and clinical medication.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to a regulatory sequence of pig liver carboxylesterase gene (PLE1 for short), and relevant bioinformatics analysis and functional verification have been carried out on the regulatory sequence. Background technique [0002] Pig liver carboxylesterase (PLE), also known as pig liver esterase (pig liveresterase), is an enzyme family composed of a variety of isozymes. The unique chiral hydrolysis characteristics of PLE make it irreplaceable in the field of organic chemical synthesis (Roberti 2000), and the research on PLE for nearly a century has mainly focused on its application in organic chemical synthesis. Studies have found that PLE can hydrolyze endogenous esters and amides such as butyrylcholine and aromatic amines, so it is reasonable to believe that PLE plays an important role in physiological processes such as the metabolism of esters in pigs, suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/55
CPCC12N9/18C12Y301/01001
Inventor 石德时周琼琼刘希妍王喜亮肖运才
Owner HUAZHONG AGRI UNIV
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