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Crassostrea gigas elongation factor-1-alpha (EF-1-alpha) promoter, and recombinant vector and application thereof

A technology of EF-1 and long oyster, which is applied in the fields of genetic engineering and molecular biology, and can solve problems such as unsuccessful development

Active Publication Date: 2020-01-14
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The large number of housekeeping genes in the organism provides many options for the development of constitutive promoters, however, the development of efficient promoters suitable for oyster oysters has not yet been successful

Method used

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  • Crassostrea gigas elongation factor-1-alpha (EF-1-alpha) promoter, and recombinant vector and application thereof
  • Crassostrea gigas elongation factor-1-alpha (EF-1-alpha) promoter, and recombinant vector and application thereof
  • Crassostrea gigas elongation factor-1-alpha (EF-1-alpha) promoter, and recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Screening of promoter fragments of housekeeping genes in long oyster.

[0030] 1) According to the published long oyster genome data, six long oyster housekeeping genes, including EF-1α, ACT, 18S rRNA, 28S rRNA and RL7, were preliminarily selected, combined with the existing transcriptome data, to further screen the long oyster embryo stability For highly expressed housekeeping genes, select the EF-1α gene with the highest expression for further analysis;

[0031] 2) Specifically analyze the transcription factor binding site in the front flank sequence of the first ATG of long oyster EF-1α, and finally use the about 3500 bp flank sequence before the ATG including the first exon and the first intron as Candidate promoter regions.

Embodiment 2

[0033] Amplification of the long oyster EF-1α promoter.

[0034] 1) Genomic DNA of oyster oyster was extracted by phenol-chloroform method, specific primers were designed according to the sequence of the promoter region of the candidate EF-1α gene, and 16 bases of homologous ends of the vector pEGFP-1 were added to the upstream and downstream primers, as shown in Table 1 Show.

[0035] Table 1 Primer sequences used to amplify the promoter of Oyster EF-1α gene

[0036]

[0037] The underlined part is the homologous end of the vector pEGFP-1 with 16 bases.

[0038] 2) Using Phusion High-Fidelity DNA Polymerase from Thermo Scientific Company to perform PCR amplification using the long oyster genomic DNA as a template. The PCR system is shown in Table 2. The PCR reaction program was: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 64°C for 30s, extension at 72°C for 1min 20s, 35 cycles; extension at 72°C for 10min.

[0039] Table 2 The PCR react...

Embodiment 3

[0043] Construction of pEGFP-CgEF-1α recombinant expression vector.

[0044] 1) The plasmid pEGFP-1 was digested with restriction endonuclease BamHI from New England BioLabs, and the restriction system is shown in Table 3. The reaction program is 37°C, 2h.

[0045] Table 3 Reaction system of BamHI digestion plasmid pEGFP-1

[0046]

[0047]2) The linearized pEGFP-1 was recovered by gel cutting, and In-Fusion ligation (In-Fusion HD Cloning Kit, TaKaRa) was performed with the above PCR recovery product. The In-Fusion connection system is shown in Table 4, and the reaction program is 50°C, 15min.

[0048] Table 4 In-Fusion connection system

[0049]

[0050] 3) Take out Escherichia coli DH5α competent cells and store them at -80°C, place them on ice, take 10 μl of the ligation product and add them to 100 μl competent cells, mix well, place on ice for 30 minutes, heat shock at 42°C for 60-90 seconds, and place on ice On 2min. Add 500 μl of SOC medium, and culture at 150...

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Abstract

The invention discloses a crassostrea gigas elongation factor-1-alpha (EF-1-alpha) promoter as well as a recombinant vector and application thereof. The invention provides an EF-1-alpha promoter. TheEF-1-alpha promoter is a high-expression housekeeping gene promoter which can be expressed in early stage of embryo development of crassostrea gigas. The EF-1-alpha promoter is specifically expressedas follows: designing a specific primer; amplifying a fragment of the crassostrea gigas EF-1-alpha promoter; inserting the crassostrea gigas EF-1-alpha promoter into a vector containing a GFP reportergene by utilizing In-fusion connection so as to obtain a recombinant vector; and then, introducing the recombinant vector into fertilized eggs of crassostrea gigas, thereby efficiently driving expression of the GFP reporter gene in embryos and larvae of crassostrea gigas. A universal-type efficient promoter sequence for crassostrea gigas is prepared according to the invention; and thus, importanttechnical basis is provided for development of CRISPR / cas9 targeted gene editing technology.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a long oyster EF-1α promoter and its recombinant vector and application. Background technique [0002] As a worldwide distribution of marine shellfish, long oyster has important economic value. In recent years, my country's long oyster genetics and breeding work has made great progress, and new varieties of long oyster "Haida No. 1" and "Haida No. 2" have been bred successively. The methods mainly used are traditional artificial selection breeding. In recent years, the application of CRISPR / Cas9 targeted gene editing technology in animal and plant genetic improvement and breed breeding has attracted widespread attention. The method of introducing Cas9 protein + sgRNA into fertilized eggs by microinjection can achieve efficient and precise target gene editing in long oyster. However, this method is not suitable for large-scale processing of fertilized eg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/65
CPCC07K14/43504C12N15/85C12N15/65C12N2800/103A01K2207/05A01K2227/70
Inventor 李琪岳晨阳于红
Owner OCEAN UNIV OF CHINA
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