Transgenic yeast containing flounder growth hormone gene, preparation and application thereof
A growth hormone and transgenic technology, applied in the field of molecular biology technology and bioengineering, can solve problems such as disease and death, speed up the growth rate of flounder and flounder fish, and achieve the effects of promoting growth, improving feed conversion rate and low cost
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Embodiment 1
[0060] Embodiment 1: Preparation of yeast with transgenic flounder growth hormone gene:
[0061] [1] Preparation of flounder growth hormone cDNA:
[0062] Extract total RNA from flounder pituitary gland, design primers ping1 and ping2 containing enzyme cutting sites BamH I and Not I, the ping1 primer sequence has 100% homology with the nucleotide sequence shown in No. 5 sequence of the sequence table, The ping2 primer sequence has 100% homology with the nucleotide sequence shown in the No. 6 sequence of the sequence table, and the flounder growth hormone cDNA is amplified by reverse transcription polymerase chain reaction (RT-PCR) method, and sequenced Make sure it matches the expected sequence.
[0063] [2] Construction of Saccharomyces cerevisiae transformation vector:
[0064] The flounder growth hormone mature peptide cDNA was digested with BamH I and Not I and then ligated into the Escherichia coli plasmid pUC-PGK to construct the transformation vector pIPGK-GH. The Es...
Embodiment 2
[0103] Embodiment 2: Identification of the yeast with transgenic flounder growth hormone gene:
[0104] Southern hybridization detection of flounder growth hormone gene recombination integrated into Saccharomyces cerevisiae genome DNA:
[0105] The genomic DNA of yeast transfected with flounder growth hormone gene was extracted, digested with EcoR I and BglI respectively, and the cDNA of flounder growth hormone was labeled with digoxin to make probes, and then Southern hybridization was performed. The result is as figure 2 Shown, wherein, M: λ / EcoRI+HindIII; 1: Genomic DNA of untransformed Saccharomyces cerevisiae (not digested); 2: Genomic DNA of untransformed Saccharomyces cerevisiae digested by EcoR I; Genomic DNA of untransformed Saccharomyces cerevisiae; 4: Genomic DNA of transformed Saccharomyces cerevisiae (undigested); 5: Genomic DNA of transformed Saccharomyces cerevisiae digested with EcoR I; 6: Genomic DNA of transformed Saccharomyces cerevisiae digested with Bgl ...
Embodiment 3
[0106] Example 3: Stability detection of flounder growth hormone gene in Saccharomyces cerevisiae:
[0107] Saccharomyces cerevisiae containing the flounder growth hormone gene was spread on the solid medium of Minimal Dextrose Plates without tryptophan, and after the colonies grew out, two clones were randomly selected and placed on the solid medium of Minimal Dextrose Plates without tryptophan Streak culture on the top; after the colonies grow out, randomly select 2 clones and culture them on the YPD solid medium... After 50 generations of continuous passage, then randomly select 10 colonies for PCR detection, all of which have specific stripes The results showed that the flounder growth hormone gene was stable in Saccharomyces cerevisiae and would not be lost with the passage of cells.
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