Studieo On Cloning And Expression Of Differentially Expressed CDNA From Pituitary Of Sex-reversal Epinephelus Akaara

StutentrHU Guoku Advisors:Prof. Zhang Yizheng and adjunct professor Wen JianjunAbstract The man-made sex reversal and functional male fish was successfully obtained by feeding 17-α methyltestosterone on Epinephelus akaara. In this study, SMART cDNA synthesis technique and long-distance PCR were used to construct the cDNA library. Clontech PCR-Select?cDNA Subtraction Kit was used to set up two differentially expressed cDNA libraries. Five hundred and sixty cDNA clones from male, and three hundred and fifty cDNA clones from female pituitary were selected for dot blotting, respectively. Twenty-nine differentially expressed cDNA fragments from male fish pituitary were sequenced. Homology analysis showed that six of them are gonad stimulating hormone precursor a -subunit (GenBank Access ion: AY207430) with homology of 97%; one is the growth hormone precursor gene (GenBank Accession:AY207431)with high homology of 99% to that from E. awoara; the other 22 fragments are not found homology in GenBank.The 5' and 3' terminal sequence of the gonad stimulating hormone precursor a -subunit gene(GT-α ) were cloned by rapid amplification of cDNA ends (RACE) and the full-length cDNA sequence was obtained from male Epinephelus akaara pituitary.The GT-α cDNA is composed of 673 nucleotide acids, in which there is a open reading frame(ORF) from 76bp to 429bp coding for a peptide of 117 amino acid residues. Virtual Northern blot showed that GT-α gene is differentially expressed during the sex reversal in male E. akaara pituitary, because the transcription product of the gene was detected only in the male E. akaara pi tin tary, no posi 1,ivo signals found in female E. akaara pituitary. Moreover, RT-PCR analysis also suggested that the GT-α gene might be highly expressed in male E. akaara pituitary. However, there was no signal detected in heart,kidney, spleen, liver, gonad, muscle, brain. The coding region ofthe gene was amplified by PCR and cloned into pET-DsbA2.0. Then the recombinant protein was observed in SDS-PAGE analysis.