性逆转石斑鱼脑垂体差异表达基因克隆的筛选

The Screening of Differentially Expressed cDNA Clones in Pituitary of Sex-Reversal Epinephelus akaara

以 1 7 α甲基睾丸酮投喂赤点石斑鱼 (Epinephelusakaara) ,成功地获得了性逆转的有功能的雄鱼。用SMARTcDNA合成和长片段PCR技术构建了性逆转前后脑垂体cDNA文库 ;用抑制消减杂交技术建立了性反转前后抑制性差减文库。对获得的 5 60个雄鱼脑垂体PCR阳性克隆和35 0个雌鱼脑垂体PCR阳性克隆进行斑点杂交 ,共筛选到 1 0 3个差异表达cDNA片段。对其中2 9个克隆进行了测序 ,与GenBank中的已知基因序列进行同源性比较 ,发现有 6个片段为促性腺激素α亚基前体基因 (GenBank注册号 :AY2 0 7430 ) ,与同属不同种的石斑鱼促性腺激素α亚基前体基因有 97%的同源性 ;1个片段为生长激素前体 (GenBank注册号 :AY2 0 7431 ) ,与同属不同种的石斑鱼生长激素前体基因有 99%的同源性 ;其余 2

We successfully obtained the man made sex reversal and functional male fish 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 libraries.Clontech PCR Select TM cDNA Subtraction Kit was used to set up two differentially expressed gene 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 α subunit (GenBank Accession:AY207430) with homology of 97%; one is the growth hormone precursor gene (GenBank Accession:AY207431)with high homology of 99% to that from Epinephelus awoara ; the others 22 fragments are not found homology in GenBank.