石斑鱼性反转相关基因ECaM的克隆及表达特征分析  

Cloning and Characterization of a Sex-Reversal-Related Gene ECaM in Epinephelus akaara Gonads

作　　者：李尚伟[1,2] 文建军[3] 刘世贵[1] 龙章富[1] 

机构地区：[1]四川大学生命科学学院 [2]贵州大学昆虫研究所,贵阳550025 [3]电子科技大学生命科学与技术学院

出　　处：《生物化学与生物物理进展》2005年第2期147-153,共7页Progress In Biochemistry and Biophysics

基　　金："863"国家高技术研究发展计划资助项目(2001AA620111);国家大洋协会项目(4-2-4).~~

摘　　要：用甲睾酮片饲喂2～4龄赤点石斑鱼(Epinephelusakaara),6周后90%以上的雌鱼性逆转为功能性雄鱼. 运用抑制性差减杂交技术(SSH),结合SMART cDNA合成和RACE-PCR方法,从性反转雄鱼性腺中克隆到钙调蛋白基因(ECaM). 该基因cDNA全长为582 bp,开放阅读框长450 bp,编码的蛋白质由149个氨基酸组成,5'端非编码区74 bp,3'端非编码区58 bp. 虚拟RNA印迹表明,ECaM在性反转雄鱼性腺中表达,而在正常雌鱼性腺中表达微弱. 各种组织的半定量RT-PCR显示,ECaM在脑、心、肝、脾、肾都有转录,在精巢和下丘脑中表达水平较高,而在肌肉中表达甚弱. 性反转不同时期性腺的半定量RT-PCR及蛋白质印迹表明,性逆转过程中性腺里ECaM的表达量逐渐增加. 上述结果提示钙调蛋白可能在赤点石斑鱼性逆转过程中发挥着作用,ECaM可能是促使石斑鱼由雌向雄转变的重要功能基因之一.Red grouper (Epinephelus akaara) females between two and four years of age were successfully reversed to functional males by feeding 17alpha-methyltestosterone (17alpha-MT) over 42 days. A gene, called ECaM, was cloned from sex-reversed male gonads by using the combinative methods of suppressive subtraction hybridization (SSH), SMART cDNA synthesis and RACE-PCR. The full-length cDNA of ECaM is 582 bp, containing a 450 bp open reading frame that encodes a 149 amino acid protein. It has a 5' untranslated region (UTR) of 74 nt and a 3' UTR of 58 nt. Virtual Northern blotting shows that ECaM is transcribed in sex-reversed male gonads but only slightly transcribed in normal female gonads. Semi-quantitative RT-PCR analyses from various tissues indicate that mRNA of ECaM can be detected in the brain, heart, liver, spleen, and kidney but slightly in the muscle. Semi-quantitative RT-PCR and Western blotting from gonads during various stages of sex reversal reveal that expression levels of ECaM in gonads increase gradually during the transformation from female to male. Calmodulin is highly conserved across species. ECaM is one of the important genes that impels grouper sex reversal.

关 键 词：石斑鱼 钙调蛋白 性腺 性反转 抑制性差减杂交 

分 类 号：Q5[生物学—生物化学] S93[农业科学—渔业资源]