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Regulatory sequence of flounder primordial germ cell Nanos3 gene and applications thereof

A primordial germ cell and regulatory sequence technology, which is applied in the field of the regulatory sequence of the primordial germ cell Nanos3 gene of flounder, can solve the problem that the specific gene markers of PGCs are not obtained and so on.

Inactive Publication Date: 2014-10-29
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports on PGCs of flounder, and PGCs-specific genes and markers of PGCs have not been obtained.

Method used

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  • Regulatory sequence of flounder primordial germ cell Nanos3 gene and applications thereof
  • Regulatory sequence of flounder primordial germ cell Nanos3 gene and applications thereof
  • Regulatory sequence of flounder primordial germ cell Nanos3 gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1. RNA extraction and SMATer3'RACE library construction

[0018] The total RNA of flounder gonads was extracted with Trizol (Invitrogen), and the specific steps were as follows: Take about 0.1 mg of flounder gonads, add 0.1 mL Trizol, mix thoroughly, then add 0.9 mL Trizol, and place at room temperature for 5 min. Add 0.2 mL of chloroform, shake vigorously for 15 s, and place at room temperature for 3 min. 12000×g, 4°C, centrifuge for 15 minutes, and carefully take out the centrifuge tube after centrifugation. Take the supernatant into a new centrifuge tube, add an equal volume of isopropanol, mix well, place at room temperature for 10 min, centrifuge at 12000×g, 4°C for 10 min. Remove the isopropanol, add 1 mL of pre-cooled 75% ethanol to wash the precipitate, centrifuge at 7500×g, 4°C for 5 min. Remove the ethanol and place it in an ultra-clean bench to volatilize the ethanol. Add 30 μL of water (RNase-free), bathe at 55-60°C for 10 minutes to dissolve the RNA, tak...

Embodiment 2

[0047] The flounder Nanos3 gene can drive the specific expression of GFP:

[0048] In this example, the Nanos3 3'UTR was amplified by PCR (removing 710-737, which is the polyadenylic acid tail), and then inserted between the reporter gene GFP and PolyA in the GFP / pSP64-polyA vector by homologous recombination.

[0049] A fragment containing GFP, Nanos3 3'UTR (removing the polyadenylic acid tail) and poly A was obtained by PCR amplification, and RNA was synthesized in vitro after purification of the fragment.

[0050] The synthetic RNA was injected into zebrafish embryos at the 1-2 cell stage, and observed under a fluorescence microscope ( image 3 ), GFP expression was found in PGCs.

[0051] Specifically:

[0052] 1. Construction of GFP / pSP64-polyA

[0053] The primers used in PCR are as follows: GFP-pSP64-5:5’-AAGCTTGGGCTGCAG GTCGACATGAGTAAAGG AGAAGAA-3’; GFP-pSP64-3:5’-TGGGAGCTCGCCCGGGGATCCCTATTTGTATAGTTCATC-3’

[0054] Use endonucleases to linearize pSP64-polyA enzyme ...

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Abstract

The invention relates to a regulator sequence of flounder primordial germ cells, and specifically relates to a regulatory sequence of a flounder primordial germ cell Nanos3 gene and applications thereof. The 3'UTR sequence of the flounder Nanos3 gene is represented by the SEQ ID No.1. The nucleotide sequence represented by the SEQ ID No.1 regulates the target gene's specific expression in primordial germ cells. The gene sequence is a specific sequence of flounder, can be used to label PGCs of marine fishes such as flounder, zebra fish, and the like, and can specifically express target genes in PCGs.

Description

technical field [0001] The invention relates to a regulatory sequence of a primordial germ cell of flounder, specifically a regulatory sequence of a Nanos3 gene of a primordial germ cell of a flounder and an application thereof. Background technique [0002] Flounder, commonly known as tooth slices, is a flounder fish, which belongs to the order Pleurotus, family Flounder, and genus Flounder. It is an important marine cultured fish in my country, Japan, South Korea and other countries. Aiming at the phenomenon that the germplasm of cultured flounder is degraded and the disease resistance is reduced, which seriously restricts the development of its breeding industry, various breeding methods have been tried in succession in recent years. Among them, sex-controlled breeding, polyploidy breeding, and molecular marker-assisted selection breeding are commonly used breeding methods, which are time-consuming and require large water bodies, and cannot introduce exogenous genes for g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
Inventor 谭训刚李美洁王倩尤锋焦爽
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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