A kind of preparation method of translysozyme gene tilapia
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
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Embodiment 1
[0025] Example 1: Construction of transgenic donor plasmid pTgf2-Hsp70-C3
[0026] Using the Oria tilapia cDNA as a template, lysozyme C3-F ( Age I) and C3-R ( Cla I) As primers, use PrimeSTAR HS DNA Polymerase (TAKARA Company) to amplify the lysozyme C3 gene by PCR. After purification and recovery of the band, it was cloned into the vector pUM19-T plasmid, and the recombinant pUM19-T-C3 was obtained by enzyme digestion and sequencing identification. plasmid. then use Age I and Cla Ⅰ Double-enzyme digestion of the pUM19-T-C3 recombinant plasmid to recover the C3 target fragment.
[0027] Restriction endonucleases for the pTgf2-Hsp70-eGFP plasmid previously constructed and preserved in the laboratory Age I and Cla I double-enzyme digestion was performed to excise the eGFP fragment in the pTgf2-Hsp70-eGFP plasmid, and the vector backbone fragment was recovered. The obtained vector backbone fragment and the recovered C3 target fragment were ligated with T4 ligase, t...
Embodiment 2
[0032] Example 2: Acquisition of Nile Tilapia Fertilized Eggs
[0033]It is very difficult to obtain freshly fertilized eggs of Nile tilapia. In order to obtain 1-cell fertilized eggs of Nile tilapia for microinjection, artificial insemination of tilapia is required. First, the robust and mature male and female tilapia were raised in indoor glass tanks, and the male and female tilapia were separated by baffles. The indoor temperature is controlled at 25-30 ℃, and the light and night time are manually controlled, with 14 hours of light and 10 hours of darkness per day. And 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 check whether there are eggs in the mouth of female tilapia. Note the time of each spawning time of the female tilapia to find the spawning cycle of the female tilapia. When the spawning cycle is reached, the ...
Embodiment 3
[0035] Example 3: Microinjection of Nile tilapia zygotes
[0036] By microinjection, the lysozyme gene Tgf 2 The transposon plasmid pTgf2-Hsp70-C3 (such as figure 1 shown) and transposase mRNA were mixed in 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 100 ng / 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 introduction volume is 2nl. After the injection of the fertilized eggs into the animal poles is completed, the fertilized eggs are placed in a special incubator for incubation at 25-28 °C, and dead embryos are regularly picked out. After hatching, the F of the translysozyme C3 gene was obtained 0 Substitute tilapia.
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