| Summary: | 碩士 === 國立臺灣大學 === 畜產學研究所 === 88 === Abstract
In this study, we used a carp pit-1 gene DNA fragment containg POU domain as probe to screen zebrafish POU family gene. We successfully obtained a POU-domain containg gene, oct-1, named clone 3-1 from zebrafish brain DNA library. Computer comparison of the sequence indicated that this sequence was identical to zebrafish oct-1 gene. By using this partcial oct-1 DNA fragment as probe to screen the zebrafish 5''-stretch plus cDNA library, we got two clone 11-2 (5''flanking ) and 8-1 (3''flanking) containing 1058bp and 2800bp, respectively.
We used PCR mediated ligation method to obtain full-length oct-1 cDNA and detected the oct-1 expression patterns in different zebrafishembryonic stages by whole-mount in situ hybridization. The results suggest that oct-1 presents in one-cell stage, through 2-cell, 4-cell, 8-cell, 16-cell, 32-cell,64-cell,128-cell, 1 k cell, 2 k cell, High, Oblong, sphere, Epiboly and Shield stage . The oct-1 gene strongly expressed in brain、neural tube、notochord and somite at prime 5 ( 24 hr )stage. The oct-1 gene still strongly expressed in brain、neural tube, but weakly in notochord and somite at prime 11 ( 30 hr )stage. In Protruding-month ( 72 hr )and upright ( 96 hr )stages, we can’t detect oct-1 mRNA in notochord and somite, but the oct-1 gene still expressed in brain and neural tube.
To express recombinant Oct-1 protein and to confirm the Oct-1 molecular weight, we subclone the oct-1 gene into pET-20 b(+) overexpressed in E.coli by IPTG induction. The recombinant Oct-1 protein can be expressed by 3 mM IPTG induction for 16hr , and the molecular weight of the insoluble recombinant Oct-1 protein is approximately 66 kDa.
With the probe prepared from the oct-1 cDNA clone 11-2 by EcoRI and SacII digestion, we identified a 9Kb oct-1 upstream DNA fragment named 2-1. Now,we have sequenced 3212 bp in the oct-1 upstream region. There are 14 TAAT sites and 1 CCAAT site in this sequence.
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