Cloning And Sequence Analysis Of Keratin 18 Gene And Promoter And Research Of Transgenic Technology In Fish Gobiocypris Rarus

China is the major Fisheries country in the world .The introduction of modern biotechnology such as transgenic technology in the traditional aquaculture has become an inevitabale trend .Being improved the living strandard,people nowadays need more requirements in the quality of lives.To fish,people are no more merely satisfied just eating as they used to be ,they need enjoying.Thus,a new industry,aquarium industry,is rising quietly.with the study of the tissue-specific promoter,the method of using fluorescent recombinant to construct fluorescent transgenic ornamental fish has become possible.Gobiocypris rarus is a unique kind of small Cyprinids, and as a new model of experimental fish,it was used in fish diseases,genetics environmental science and other fields of study. Gobiocypris rarus has the potential to develop into an ornamental fish like zabrafish(Danio Rerio),medaka(Oryzias latipes)by use of the Transgenic techniques.Cytokeratin 18 (K18) is an'S'keratin and is the first of the intermediate filament proteins to be expressed during mammalian and amphibian development. According to the published conserved DNA sequences of keratin 18 from human, xenopus and zebrafish ,two pairs of primers were designed to obtain 2779bp of keratin 18 gene derived from the genome DNA of Gobiocypris rarus by PCR. And then through the genome walking upstream and downstream sequences were also amplificated. And then the full amplifiated fragment of keratin 18 was inserted into Escherichia coli cloning vector pMD19-T and sequenced. The result indicated that the keratin 18 of Gobiocypris rarus was successfully obtained about 3765 bp. The analysis of NCBI's blastn showed that comparing Gobiocypris rarus with zebrafish,the keratin 18 DNA sequence was high conserved with 73.6% similarity. Finally, biologically analysis and ORF prediction showed that the keratin 18 DNA sequence contained 7 exons and 6 introns, and coded for a protein with 367 amino acids.Transposon is the basic unit of self - rep lication and shift in DNA, it exists in all fields of biology, transposon and its related technology are the powerfulweapons for studying the function of the genome in the post - genome era.The 1223 bp promoter region fragment was inserted into red fluorescent protein gene vector and transposase enzyme gene vector.The recombinants was then injected into the fertilized eggs of Gobiocypris rarus by the method of micro-injection.the red fluorescent could be observed by micro fluoroscope.This study proved that the keratin 18 promoter separated had effective transcription activity.