Injection for knocking down expression of 14-3-3 gene of turbot and use method

A technology for gene expression, turbot, applied in the field of molecular biology

Pending Publication Date: 2019-08-23
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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Problems solved by technology

The research in aquatic animals is mainly focused on the model animal zebrafish. The research method is mainly to inject dsRNA in the embryo for interference, and to study the role of genes in the growth and development process. However, the injection of interference in the juvenile muscle The research method has not been reported

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  • Injection for knocking down expression of 14-3-3 gene of turbot and use method
  • Injection for knocking down expression of 14-3-3 gene of turbot and use method
  • Injection for knocking down expression of 14-3-3 gene of turbot and use method

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Embodiment 1

[0017] Injection for knocking down the expression of turbot 14-3-3 gene and its use method, the specific steps are as follows: preparation of turbot 14-3-3 gene dsRNA, intramuscular injection interference experiment and verification of interference effect

[0018] 1. RNA extraction from main expression tissue of 14-3-3 gene: Collect gill tissue, use Tiangen RNA rapid extraction kit for RNA extraction, detect RNA quality by 1% agarose gel electrophoresis, and measure RNA concentration by Nanodrop2000 UV spectrophotometer , Store at -80°C after passing the test.

[0019] 2. Full-length cDNA cloning of the 14-3-3 gene: Reverse transcribe the gill tissue RNA into cDNA, obtain the full-length cDNA sequence by TA cloning and RACE methods, and perform bioinformatics analysis on the sequence, such as the coding region predictions, etc.

[0020] 3. Design of dsRNA primers: Based on the full-length cDNA sequence of the turbot 14-3-3 gene, 2 pairs of primers for the preparation of doubl...

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Abstract

The invention relates to an injection for knocking down the expression of 14-3-3 gene of a turbot and a use method, and belongs to the field of molecular biology. The preparation method of the injection includes the following steps of (1) RNA extraction of gene main expression tissue, and (2) preparation of dsRNA, wherein specific primers are designed to synthesize a DNA product fragment accordingto the CDS sequence of the target gene. A target DNA fragment is obtained through a PCR with the cDNA synthesized by reverse transcription of the RNA extracted in the step (1) as a template, a DNA product is recovered, the dsRNA is synthesized with the recovered product as a template, and the synthesized dsRNA is diluted with physiological saline to reach an effective concentration of 4 microgram/gram and injected into the back muscles of the fish by 2-4 injections by multiple-point injection. The injection makes direct gene knockdown at the fish individual level to facilitate the study of the function of the 14-3-3 gene of the turbot.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a dsRNA injection for knocking down turbot 14-3-3 gene expression and a usage method. Background technique [0002] RNA interference (RNA interference, RNAi) refers to highly conserved in the evolutionary process, induced by double-stranded RNA (double-stranded RNA, dsRNA), through the inhibition of mRNA translation or the efficient and specific degradation of mRNA to mediate post-transcriptional gene silencing phenomenon. The process of gene silencing mediated by dsRNA usually needs to go through three steps. Firstly, after dsRNA enters the cell, it combines with the enzyme protein Dicer and is cut into small fragments of 21-23nt by Dicer. These small fragments are called siRNA; Helicase, etc.) combine to form the RNA-induced silencing complex (RNA-induced silencing complex, RISC); the last activated RISC binds to the target mRNA under the guidance of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89C12N15/113
CPCC12N15/89C12N15/113C12N2310/14
Inventor 刘志峰马爱军张金生赵亭亭杨双双杨敬昆曲江波
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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