Breeding method of albino paramisgurnus dabryanus capable of being inherited stably

An albino technology of paraloach maxima, which is applied to the introduction of foreign genetic material using vectors, recombinant DNA technology, animal husbandry, etc., can solve the problems of supply and demand difficulties of albino fish species, and achieve the effect of high knockout efficiency and short cycle.

Inactive Publication Date: 2017-05-10
HUAZHONG AGRI UNIV
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Problems solved by technology

It is used for directional modification of fish body color, which can not only effectively solve the problem of difficult supply a

Method used

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  • Breeding method of albino paramisgurnus dabryanus capable of being inherited stably
  • Breeding method of albino paramisgurnus dabryanus capable of being inherited stably
  • Breeding method of albino paramisgurnus dabryanus capable of being inherited stably

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Embodiment Construction

[0030] The implementation and application of the present invention will be described below in conjunction with implementation cases.

[0031] 1. Extraction of total RNA from the skin of Paraloach grandiflora

[0032] Use TaKaRa's RNAiso Plus reagent to extract the total RNA of Paraloach macronata. The specific steps are as follows:

[0033] 1) RNA extraction: all utensils, surgical scissors, and tweezers should be treated with DEPC overnight; the water used is RNase-free Water purchased by TaKaRa; the centrifuge EP tube, each type of pipette tip is dedicated for RNA extraction or DEPC water Soak overnight and autoclave. Note: Wear a mask, change gloves, and avoid talking during the extraction of total RNA. Pre-cool the sampling instruments on ice before the experiment;

[0034] 2) Put to death the Paraloach, quickly separate the skin tissue, and take 30mg-50mg tissue sample in a 2mL centrifuge tube, place the centrifuge tube in an ice bath, add 1.5mL RNAiso Plus reagent beforehand, ...

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Abstract

The invention discloses a breeding method of albino paramisgurnus dabryanus capable of being inherited stably. The method comprises the steps as follows: a TYR (tyrosinase) gene target site is designed on a TYR gene sequence of paramisgurnus dabryanus, then an upstream primer and a downstream primer matched with the upstream primer are designed, PCR (polymerase chain reaction) amplification is performed with framework plasmid as a template, in-vitro transcription and purification are performed, and gRNA is obtained; Cas9 mRNA is obtained from linear Cas9 plasmid as a template after in-vitro transcription and purification; micro-injection is performed on fertilized eggs of the paramisgurnus dabryanus, the fertilized eggs are then incubated, gene knock-out fish is subjected to hybridization, and the albino paramisgurnus dabryanus capable of being inherited stably is obtained.

Description

Technical field [0001] The present invention belongs to the field of molecular breeding, and in particular relates to a method for using the CRISPR / Cas9 system to knock out the pigment gene of Paraloach macroschis to produce albino Parasquama macross. Background technique [0002] With the steady development of the ornamental aquarium industry in China, the species of ornamental creatures have increased sharply year by year, and the native fish species represented by the mackerel family are gradually becoming familiar to the majority of aquarium lovers. Among them, albino loach fish species can be seen from the appearance of white or light red skin, red and transparent eyes. Because of the singularity and rarity of this trait, it is especially sought after by the majority of aquarium consumers. At present, these new ornamental fish resources are mainly harvested and screened in the wild, with high randomness and very low output, which can hardly meet the curiosity needs of the m...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
Inventor 曹小娟徐秀文高坚
Owner HUAZHONG AGRI UNIV
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