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DsRNA of oncorhynchus mykiss ccne1 gene and application thereof

A gene and rainbow trout technology, applied in the dsRNA of rainbow trout ccne1 gene and its application field, can solve the problems of product performance decline and stress tolerance reduction

Pending Publication Date: 2022-08-09
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, during the production, it was found that the performance of the product caused by gonad development and the reduction of stress tolerance have caused adverse effects on production and breeding.

Method used

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  • DsRNA of oncorhynchus mykiss ccne1 gene and application thereof
  • DsRNA of oncorhynchus mykiss ccne1 gene and application thereof
  • DsRNA of oncorhynchus mykiss ccne1 gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0027] The cloning of embodiment 1 rainbow trout ccne1 gene fragment

[0028] 1. Total RNA extraction

[0029] The juvenile rainbow trout (body weight 30±5g) was used in the experiment. After MS222 anesthesia, the intact glandular tissue was collected on the ice tray, and the samples were collected in liquid nitrogen. The total RNA of the rainbow trout gonadal tissue was extracted by the kit method. The specific method refers to Simply P total RNA extraction. Kit (Bio Flux) instructions. RNA concentration and purity were checked using a UV spectrophotometer (Analytikjena, Germany).

[0030] 2. Reverse transcription of cDNA

[0031] The total RNA was reverse transcribed, and cDNA was obtained by reverse transcription using the reverse transcription kit PrimeScript RT Reagent Kit (TaKaRa, Dalian). The reverse transcription system was: total RNA 1 μg, Oligo dT Primer 1 μL, dNTP Mixture 1 μL, RNAase- free dH 2 0 to 10 μL. After incubation at 65°C for 5 min, it was rapidly coo...

Embodiment 2

[0046] Example 2 The interference gene fragment of rainbow trout ccne1 gene and the synthesis of dsRNA

[0047] Based on the rainbow trout ccne1 gene whose nucleotide sequence is shown in SEQ ID NO.1, use https: / / www.dkfz.de / signaling / e-rnai3 / website to design the interference gene fragment primers of the rainbow trout ccne1 gene, above, The T7 sequence needs to be added before the downstream primer. See Table 2 for primer information.

[0048] Use the upstream primer SEQ ID NO.6 and the downstream primer SEQ ID NO.7 containing the T7 promoter to obtain a PCR product through PCR amplification, the sequence is such as SEQ ID NO.3, which is purified and recovered by a PCR purification kit to obtain rainbow trout ccne1 Interfering gene fragments of genes.

[0049] Table 2

[0050]

[0051] The sequence of the interference gene fragment of the rainbow trout ccne1 gene is shown in SEQ ID NO.3.

[0052] SEQ ID NO. 3:

[0053] CCAGTGTGGAGCTTTGGTTTGCTGGAATCCTGAGTCGGTTCACACCATC...

Embodiment 3

[0060] Embodiment 3 Rainbow trout ccne1 gene silencing efficiency detection

[0061] 1. Injection of rainbow trout dsRNA

[0062] Using a 1 mL syringe, 50 μl of PBS containing 30 μg of dsRNA was injected into the peritoneal cavity of rainbow trout. The experimental fish were divided into two groups, namely the experimental group and the control group, with 10 fish in each group. In the experimental group, 50 μl of PBS solution containing dsRNA (concentration of 0.6 μg / μl) was injected into juvenile rainbow trout (body) with a 1 mL syringe. The mass is 20g±1.2g) intraperitoneal cavity, and the control group was injected with an equal volume of 1×PBS solution as a control for three consecutive days, and the gonad tissue was collected 72h after the last injection.

[0063] 2. Changes in ccne1 gene expression

[0064] The total RNA of gonad tissue was extracted 72h after injection, and cDNA was obtained by reverse transcription, which was used as a template to detect the relativ...

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Abstract

The invention discloses dsRNA of oncorhynchus mykiss ccne1 gene and application of the dsRNA, and belongs to the technical field of biology. The invention provides a rainbow trout ccne1 gene, an interference gene segment and dsRNA of the rainbow trout ccne1 gene. The method comprises the following steps: firstly, cloning by taking total RNA (Ribonucleic Acid) of gonad tissues of juvenile rainbow trout as a template to obtain a gene sequence of ccne1, wherein the nucleotide sequence of the gene sequence is SEQ ID NO.1, and the amino acid sequence of the gene sequence is SEQ ID NO.2; a ccne1 gene sequence is used as a template to design a primer of an interference gene segment, the interference gene segment is obtained, the nucleotide sequence is SEQ ID NO.3, and the dsRNA of the rainbow trout ccne1 gene is synthesized through in-vitro transcription. The dsRNA of the oncorhynchus mykiss ccne1 gene can efficiently silence the ccne1 gene and effectively reduce the expression of other meiosis related genes in oncorhynchus mykiss, and provides a research idea for oncorhynchus mykiss meiosis pathway analysis.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the dsRNA of rainbow trout ccne1 gene and its application. Background technique [0002] Rainbow trout (Oncorhynchus mykiss) belongs to the order Salmonoides, Salmonidae, Salmonidae, Salmon genus, and is named after the band-like red stripes on the side of the body. Rainbow trout, as the main cultured species of salmon trout in my country, is loved by consumers for its delicate meat and delicious taste, and has high commercial value. However, in the production, it was found that the decline of product performance and the reduction of stress tolerance caused by the development of the gonads caused the adverse effects of production and breeding. In recent years, the key scientific issue of gonadal differentiation in rainbow trout has become a research hotspot, and the analysis of its key regulatory molecular mechanisms has been explored. [0003] RNAi (RNAinterference, RNAi) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/89C12N15/12
CPCC12N15/113C12N15/89C07K14/461C12N2310/14Y02A40/81
Inventor 黄天晴刘恩慧徐革锋谷伟王高超王炳谦
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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