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A method and application of germplasm construction of marine flounder and flounder based on genome editing

A genome editing and construction method technology, applied in the field of marine fish genetics and breeding, can solve problems such as gonadal development, biological sex and fish size effects

Active Publication Date: 2017-07-14
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether the gene knockout (mutation) will affect the gonad development, physiological sex and fish body size has not been reported at home and abroad.

Method used

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  • A method and application of germplasm construction of marine flounder and flounder based on genome editing
  • A method and application of germplasm construction of marine flounder and flounder based on genome editing
  • A method and application of germplasm construction of marine flounder and flounder based on genome editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Construction of TALEN plasmids for genome editing of Dmrt1 in half-smooth tongue sole:

[0031] 1. Construction method of genome editing TALEN plasmid:

[0032] According to the genome sequence SEQ ID NO: 1 of the half-smooth tongue sole Dmrt1 gene, a pair of TALEN binding sites were selected in the interval 80-140 nucleotides downstream of the start codon ATG in the coding region of exon 1, and the length of each binding site was 16-18bp nucleotides, the left and right binding sites are separated by 15-17bp, the sequence of the left binding site is 5'-CCCGCTGCAGGAACCAC-3'(SEQ ID NO:4); the sequence of the right binding site is 5'- AGCGTTTGTGGCCCTTC-3' (SEQ ID NO: 5); the upstream base of the starting base at the 5' end of the binding site sequence should be T.

[0033] The main steps of constructing the TALEN plasmid are as follows: first, according to the selected 17-base TALEN binding site sequence ( figure 1), using Addgene's kit TALEN Golden Gate toolk...

Embodiment 2

[0046] Example 2. In vitro transcription of genome editing plasmids:

[0047] The genome editing (TALEN) plasmid was transformed into Escherichia coli and cultured, and the genome editing plasmid was extracted using a plasmid extraction kit; the genome editing plasmid was digested with the corresponding restriction endonuclease NotI to make it linear; commercially sourced The in vitro transcription kit transcribes the linearized plasmid into the corresponding mRNA and purifies it.

[0048] The following takes the in vitro transcription of the Dmrt1 genome editing (TALEN) plasmid in half-smooth tongue sole as an example to describe in detail.

[0049] 1) The Dmrt1 TALEN plasmid required for in vitro transcription was obtained in two ways.

[0050] (1) Expansion culture of tongue sole Dmrt1 TALEN stored at -80℃

[0051] 1. Take out the strains stored at -80°C from the ultra-low temperature refrigerator, inoculate them into 100ml of LB medium after thawing, and incubate for 12 ...

Embodiment 3

[0078] Example 3, transfer of in vitro transcribed mRNA to fertilized eggs of marine flounder and flounder and culture of fertilized eggs

[0079] Adjust the concentration of the purified TALEN mRNA to 90-110ng / ul; collect the fertilized eggs of flounder and flounder with normal development at the 1-4 cell stage; transfer 100-300pg of TALEN mRNA into the animal pole of the fertilized eggs by conventional methods , then put the fertilized eggs into a beaker filled with sterile seawater at a constant temperature (22°C-23°C for half-smooth tongue sole, 14°C-16°C for flounder, and 12°C-13°C for turbot), And add 10000x 1% methylene blue solution (2ul per 100ml sterile seawater) into the beaker, and transfer the surviving fertilized eggs into new sterile seawater added with methylene blue every 3-5h. The cultivation method improves the hatching rate by 5%-10% compared with the traditional hatching pond flowing water culture.

[0080] The following takes the transfer of Dmrt1 TALEN ...

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Abstract

The invention provides a genome-editing-based seawater flounder germplasm building method. Take cynoglossus semilaevis Dmrt1 genes for example, the genome-edited TALEN plasmids of the Dmrt1 genes are built, the genome-edited TALEN plasmids are transcribed into mRNA in vitro and transferred to the fertilized egg animal pole of cynoglossus semilaevis 1-4 cell stages, the fertilized eggs are cultivated into adult fish, and a PCR method is used to screen out gene-mutated F0 generation fish so as to build the adult fish with gene site-directed mutation. The invention further provides a method for cultivating the new germplasm of seawater fish with the gene site-directed mutation, and the method is characterized in that the F0 generation fish is hybridized with wild fish to obtain hybrid filial generation fish, genetic mutation type F0 generation fish is detected to serve as male and female parents to perform artificial reproduction, F1 generation fish with gene double-site mutation is screened out, and further hybridization is performed to obtain homozygous mutation system F2 generation fish. The method has the advantages that site-directed mutation of the flounder genes can be achieved, and a new method is provided for the flounder gene function researches and new germplasm creating.

Description

technical field [0001] The invention belongs to the technical field of marine fish genetics and breeding, and specifically relates to a method for constructing a new germplasm of marine flounder fish through genome editing, that is, performing site-directed mutation on a specific gene in the genome of marine flounder fish such as semi-smooth tongue sole and cultivating it into an adult way of fish. Background technique [0002] Genome editing technology is a gene manipulation technology developed in recent years for targeted modification of the genome. It can knock out any gene or sequence in the genome or introduce foreign genes into specific sites in the genome. Genome editing mainly includes TALEN technology and CRISPR / Cas9 technology. Genome editing technology is of great significance and application value in the analysis of gene function and genetic engineering breeding of marine fish. Carrying out research on genome editing technology of marine flounder fish is of gre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90A01K61/10A01K67/027
CPCA01K61/00A01K67/0275A01K2227/40A01K2267/02C07K14/461C12N15/87Y02A40/81
Inventor 陈松林崔忠凯郑汉其刘云王娜林帆王文文张宁董忠典李仰真
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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