性逆转石斑鱼性腺差异表达基因的克隆和筛选

以 17α 甲基睾丸酮 (17α MT)饲喂 2～ 4龄赤点石斑鱼 (Epinephelusakaara) ,成功地促使其性转变为具有生殖功能的雄鱼 .应用抑制性差减杂交 (SSH)技术构建了石斑鱼性反转前后性腺组织的SMARTcDNA文库及其cDNA差减文库 ,从中随机挑取 12 0 0个克隆进行了PCR和斑点杂交筛选 ,得到 12 0个差异表达cDNA片段 .挑选 71个cDNA克隆测序 ,将所测序列经GenBank检索和生物信息学比较 ,发现有 5 1个cDNA片段序列无明显的同源性 ,2 0个片段与报道的基因有较高同源性 .在这 2 0个具同源性的片段中有 3个片段可能是与性别分化密切相关的重要功能基因 ,它们是钙调蛋白基因、活性蛋白激酶C受体基因和一氧化氮合酶蛋白抑制剂基因 .这 3个基因被分别命名为鱼钙调蛋白基因 (GenBankaccession :AY2 8136 3)、鱼活性蛋白激酶C受体基因 (GenBankaccession :AY2 8136 4)和鱼一氧化氮合酶蛋白抑制剂基因 (GenBankaccession :AY2 8136 5 ) .

Cloning and Screening of Differentially Expressed Genes from Sex-Reversed Gonad of Epinephelus akaara

Two to four year old females of red spotted grouper ( Epinephelus akaara ) were successfully reversed into functional males by means of feeding 17α methyltestosterone (17α MT) at the dosage of 5×10 -6 (W/W) over a 60 days duration. A systematic study was initiated to identify differentially expressed genes in the process of sex reversal of the grouper by using suppression subtractive hybridization (SSH) technique. Some differentially expressed genes between female gonad (FG) and male gonad (MG) in the grouper were obtained. Two SSH plasmid libraries for gonads of pre reversed and post reversed grouper were constructed. And then differentially expressed genes were screened by PCR and dot blotting. 800 MG positive clones and 400 FG positive clones were obtained by PCR amplification, and 520 MG and 250 FG PCR positive clones were selected to carry on dot blotting. 51 MG and 20 FG dot blotting positive clones were cloned and sequenced. Searching GenBank by using these nucleotide sequences indicated that 51 cDNA fragments could not find their homologous sequences in the database. Of the 20 known genes, 3 may be closely relevant to sex reversal of the grouper according to the reported papers, and they are calmodulin ( CAL1 ), receptor for activated protein kinase C ( RACK1 ) and protein inhibitor of neuronal nitric oxide synthase ( PIN ), respectively. They were designated ECAL1 (GenBank accession:AY281363), ERACK1 (GenBank accession: AY281364)and EPIN (GenBank accession: AY281365), respectively.