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Preparation method of transgenic tilapia with lysozyme gene

A technology of tilapia and lysozyme, applied in the field of transgenics, can solve the problems of limited preparation of transgenic tilapias, achieve the effect of improving immunity and broadening the research space

Inactive Publication Date: 2014-07-23
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the difficulty in obtaining freshly fertilized tilapia roe in vitro in the prior art, which leads to the limited technical defect in the preparation of transgenic tilapia, the present invention provides a method for preparing transgenic tilapia

Method used

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  • Preparation method of transgenic tilapia with lysozyme gene
  • Preparation method of transgenic tilapia with lysozyme gene
  • Preparation method of transgenic tilapia with lysozyme gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of transgene donor plasmid pTgf2-Hsp70-C3

[0026] Using the cDNA of Oria tilapia as a template, lysozyme C3-F ( Age I) and C3-R ( cla I) As a primer, use PrimeSTAR HS DNA Polymerase (TAKARA Company) to amplify the lysozyme C3 gene by PCR. After the band is purified and recovered, it is cloned into the vector pUM19-T plasmid, digested and sequenced to identify the pUM19-T-C3 recombinant plasmid. then use Age I and cla Ⅰ Digest the recombinant plasmid pUM19-T-C3 with double restriction enzymes, and recover the C3 target fragment.

[0027] Use restriction endonucleases on the pTgf2-Hsp70-eGFP plasmid previously constructed in the laboratory Age I and cla I performed double enzyme digestion, excised the eGFP fragment in the pTgf2-Hsp70-eGFP plasmid, and recovered the vector backbone fragment. Ligate the obtained vector backbone fragment and the recovered C3 target fragment with T4 ligase, transform into competent Escherichia coli DH5α,...

Embodiment 2

[0032] Embodiment 2: Obtaining of fertilized eggs of Nile tilapia

[0033]It is very difficult to obtain freshly fertilized eggs of Nile tilapia. In order to obtain fertilized eggs at the 1-cell stage of Nile tilapia for microinjection, artificial fertilization of tilapia is required. First put the robust and mature male and female tilapias into indoor glass tanks to raise respectively, and separate the male and female tilapias with baffles. The indoor temperature was controlled at 25-30°C, and the time of light and night was artificially controlled, with 14 hours of light and 10 hours of darkness every day. Moreover, in the feed of tilapia, vitamin E is fed twice a week to increase the reproductive capacity of tilapia.

[0034] Focus on observing the reproductive activities of female fish every day, and regularly detect whether there are eggs in the mouth of female tilapia. Note down the time of each spawning of the female tilapia to find the spawning cycle of the female ti...

Embodiment 3

[0035] Example 3: Microinjection of fertilized eggs of Nile tilapia

[0036] Using microinjection method, will carry lysozyme gene Tgf 2 Transposon plasmid pTgf2-Hsp70-C3 (such as figure 1 shown) and transposase mRNA were mixed at a ratio of 1:2, Tgf 2 The concentration of transposon plasmid pTgf2-Hsp70-C3 is 50ng / μl, and the concentration of mRNA encoding transposase is 100ng / μl, and then microinjected into fertilized eggs within 1 hour of tilapia spawning , the introduction site is the animal pole of the fertilized egg, and the total volume of introduction is 2nl. After the animal pole injection of fertilized eggs is completed, the fertilized eggs are placed in a special incubator for incubation, incubated at 25-28°C, and dead embryos are regularly picked out. Immediately after hatching, F 0 Substitute tilapia.

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Abstract

The invention belongs to the technical field of transgenosis and in particular discloses a preparation method of transgenic tilapia with a lysozyme gene. A new tilapia strain which can achieve induced expression of tilapia lysozyme C3 is obtained by introducing a Tgf2 transposon carrying a lysozyme C3 gene driven by an Hsp70 promoter into tilapia zygotes and integrating the Hsp70 promoter and the lysozyme C3 gene onto a tilapia genome by utilizing the transposition characteristic of the transposon. By adopting the preparation method, not only can the immunity of the tilapia be improved and the new tilapia variety with stronger disease resistance be bred but also the Tgf2 transposon is introduced into construction of the transgenic tilapia for the first time and a new method is provided for importing exogenous genes into cultured fishes. The preparation method has great theoretical significance and application value.

Description

technical field [0001] The invention relates to the field of transgenic technology, and specifically discloses a preparation method of tilapia transgenic for lysozyme. Background technique [0002] Tilapia belongs to the order Percifomes, family Cichlidae, genus Tilapia, and is a worldwide farmed fish. However, in recent years, due to reasons such as high breeding density and deterioration of the breeding environment, streptococcal disease broke out in cultured tilapia, resulting in a large number of deaths of cultured tilapia, causing huge economic losses and threatening the healthy development of tilapia farming. . Therefore, there is a need to establish a new strain of tilapia that can effectively resist streptococcal disease. [0003] Transgenic is a fast and efficient method to improve the traits of aquaculture species. There are many methods for transgenic, and transposon transgenic technology is widely used because of its convenient operation and high integration ef...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N5/10A01K67/027
Inventor 叶星孙成飞董浚键瞿兰卢迈新
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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