不同倍性鲫鲤性腺型芳香化酶cyp19a1a基因cDNA的克隆及表达  

Molecular cloning and comparative expression patterns of cyp19a1a of gene in different ploidy cyprinid fishes

作　　者：陶敏[1] 刘少军[1] 张卓慧[1] 陈婕[1] 刘文彬[1] 刘筠[1] 

机构地区：[1]湖南师范大学生命科学学院,教育部蛋白质化学与发育生物学重点实验室,湖南长沙410081

出　　处：《水产学报》2014年第9期1201-1210,共10页Journal of Fisheries of China

基　　金：湖南省教育厅资助科研项目(14B115;13C523);湖南省自然科学基金项目(14JJ2148);国家自然科学基金重大国际合作项目(31210103918);湖南省生物发育工程及新产品研发协同创新中心(20134486)

摘　　要：为研究性腺型芳香化酶P450aromA(cyp19a1a基因编码)在不同倍性鲫鲤卵巢发育过程中的作用,实验采用同源克隆和cDNA末端快速扩增技术(RACE),获得了二倍体红鲫、三倍体湘云鲫和四倍体鲫鲤的cyp19a1a基因cDNA全长。结果显示,3种鱼cyp19a1a基因均编码517个氨基酸残基,而且编码的蛋白都包含P450aromA特有的跨膜螺旋区、I-螺旋区、Ozol’s肽区、芳香化酶特异保守区以及血红素结合区。采用RT-PCR分析cyp19a1a基因mRNA在3种不同倍性鱼类组织中的表达情况,结果显示,cyp19a1a基因主要在实验鱼的卵巢中表达,其次在精巢、脑、脾脏有少量表达。采用实时荧光定量PCR对cyp19a1a基因mRNA在不同倍性鱼卵巢中的表达进行分析,结果发现,cyp19a1a基因在不同倍性鱼的非繁殖期卵巢的表达都高于繁殖期卵巢,并且在繁殖期和非繁殖期,cyp19a1a基因在三倍体湘云鲫的表达量高于二倍体红鲫和四倍体鲫鲤。采用免疫组织化学方法对CYP19A蛋白在不同倍性鲫鲤卵巢中的定位进行分析,结果发现,CYP19A蛋白主要定位在滤泡细胞、Ⅳ时相卵母细胞的放射膜上以及Ⅱ时相卵母细胞的卵浆中。研究表明,性腺型芳香化酶在不同倍性鲫鲤卵巢发育过程中的表达存在一定的差异性,三倍体鱼cyp19a1a基因mRNA水平表达异常,推测与其不育有关联性。In order to study the role of the gonad-specific aromatase（ P450aromA） encoded by cyp19a1 a gene in ovary development of different ploidy cyprinid fishes,molecular cloning and comparative expression patterns of cyp19a1 a of gene in different ploidy cyprinid fishes were carried out. By PCR and rapid amplification of cDNA ends（ RACE）,the full length cDNAs of cyp19a1 a in diploid red crucian carp（ Carassius auratus red var.）,triploid crucian carp and allotetraploid hybrids were obtained. Our data showed that all the cDNAs of cyp19a1 a gene in the three different ploidy fishes encode 517 amino acids,which contains transmembrane helix region,I-helix region,Ozol ＇s peptide region,aromatase-specific conserved region and heme-binding region. By RT-PCR using specific primers against the same sequences of coding regions in different ploidy cyprinid fishes,a comparative study of the expression pattern of cyp19a1 a was carried out. This result showed that cyp19a1 a was preferentially expressed in ovaries of these three fishes,but also was present at much lower levels in testes,brains and spleens. The expression pattern of cyp19a1 a in ovaries of different ploidy fishes from the pre-ovulation and the ovulation was also studied by Real-time PCR,and the results showed that the expression of this gene was greatly different among the three different ploidy fishes. In the ovulation and pre-ovulation,the expression of cyp19a1 a mRNA in ovary of triploids was the highest among these three fishes. By immunohistochemistry,the CYP19 A peptide locations in the ovary of the different ploidy fishes were studied,which indicated that CYP19 A was found in follicle cells,outside layer of zona radiate of Ⅳ oocytes,and ooplasm of stage Ⅱ oocytes. These researches showed that there are different expressions of the gonad-specific aromatase during the ovary development in different ploidy cyprinid fishes. In addition,the abnormal expression of cyp19a1 a mRNA in triploids might be associated with sterility.

关 键 词：不同倍性鲫鲤 性腺型芳香化酶 卵巢发育 mRNA表达 组织定位 

分 类 号：Q786[生物学—分子生物学] S965.1[农业科学—水产养殖]