Construction,Integration And Expression Of Green Fluorescent Protein Recombination Gene In Goldfish

Green fluorescent protein (GFP) was extracted from jellyfish (Aequerea Victoria) of ocean invertebrate. GFP can emit green fluorescent when it is illuminated by light of suitable wavelength. The emissions of fluorescence need not any additional factors, such as substrate, supplementary factor. Green fluorescent protein is widely applied in researches of modern life science, such as gene product moved-process in vivo, protein localization, drug screening and preliminary selection of transgenic individual as a molecular marker. In this study, the distinctive character of green fluorescent protein was using as a screening method to improve determination efficiency of transgenic individuals, and simultaneously to provide reference concerning foreign gene integration and regulation.In the research of transgenic fish, green fluorescent protein gene was sub-cloned to downstream of carp P -actin gene promoter that was cloned in pUCUSA by molecular recombination technology. Thus pAGFP plasmid was constructed successfully. The recombination was determined by digestion of restriction enzyme and sequencing. Recombination gene was cut-down and introduced into the nuclei of oocytes or the cytoplasm of goldfish at one-cell stage via microinjection. The results as follows:(1) Fluorescence was observed from embryo under suitable UV light after microinjection 36 hours. The fluorescent ratio of Gastrula embryo period was up to 25%. During developing backward, fluorescent ratio was gradually descending. It was relatively stable after muscle effect period.(2) Goldfish zygote were transformed by different copies 10\ 10\ 10 and 10" respectively, results showed that foreign copies was not existed simple linear relativity.(3) Genomic DNA was extracted from 12 preliminary screening fish. Thetransgenic goldfish were detected and screened by PCR, Southern blot. The results showed that PCR amplify a 478bp specific molecular band in 5 individuals, Southern blot results showed a strong signal in transgenic fish. It is concluded that ' green fluorescent protein gene has been integrated and expressed in at least 2 transgenic fish, it is also showed that foreign gene integrating positions and copy numbers are differ in different individuals.