稀有鮈鲫脑芳香化酶cDNA片段的克隆与表达分析

摘要

P450 aromatase (P450arom), an enzyme catalyzing the synthesis of estrogens, is thought to play a key role in sex differentiation of neural and reproductive development in vertebrates. Most of the mammals have only one aromatase, but many teleost fish, including the zebrafish (Danio rerio) and medaka (Oryzias latipes) have two isoforms of aromatase encoded by two distinct genes, Cyp19a expressed predominantly in the ovary and Cyp19b in the brain. Gobiocypris rarus is an emerging model fish in China because of its small size, transparent embryonic biology, spawning round the year, easy breeding, short generation and sensitivity in toxicology. However, the mechanisms of sex determination and differentiation are still unclear in this fish. Aromatase as the important factor in the differentiation of sex is also unavailable, so that the exact roles of aromatase gene in neural or ovarian development in this fish are unclear. To understand the mechanisms of sex differentiation and the role of aromatase in this process, we cloned the partial sequences of Cyp19b cDNA of Gobiocypris rarus by reverse transcription-polymerase chain reaction with degenerate primers dependant on the conservative sequences of the gene in other vertebrates, and we also examined its expression pattern in the tissues of adult fish and the developmental process of embryos in this fish by gene specific primers. The partial sequence of Cyp19b of Gobiocypris rarus we cloned consisted of 1070 base pairs which encoded 357 amino acids. Structural analysis of the deduced amino acid sequence indicated that it contained three specific domains of aromatases, substrate-binding loop, distal helix I and the steroid-binding domain. Multiple alignment and phylogenetic analysis showed that this protein of Gobiocypris rarus shared 57%- 93% identities with P450arom proteins of other species and it was most similar to P450aromBs of Danio rerio, Carassius auratus and Cyprinus carpio in 91%, 92% and 93%, respectively. This result indicated that the gene we got was Cyplgb of Gobiocypris rarus. The tissue expression pattern analysis by RT-PCR with specific primers showed that Cyp19b was predominantly expressed in the brain in both sexes, and the expression in the brain of female was slightly higher in female but not obviously stronger than that in the male in statistics. It was also expressed in eye, gill, kidney, intestine of both sexes, but not detectable expressions in other tissue like heart, liver, spleen, air bladder, and muscle. Ontogenic analysis by RT-PCR with specific primers showed that no expression was detected in the zygote, and the onset of expression of Cyp19b was from blastula, then the expression reached a higher level in neurnla, after that it declined. The expression of Cyp19b burst again in 5d after hatching and it kept in a very high level till 30d after hatching. These results indicate that Cyp19b is expressed predominantly in the brain of adult fish, and it is also expressed highly in the stage of neurula. Cyp19b may play important roles in the development of central neural system and the sexual differentiation of the brain. The beginning of the expression from blastula suggest that Cyp19b is not a maternal factor for the embryos of Gobiocypris rarus.%芳香化酶(P450arom)是雌激素合成过程中的关键酶,在性别分化中起重要作用.鱼类存在卵巢性和脑型两种芳香化酶,分别由Cyp19a和Cyp19b编码.稀有鮈鲫作为我国特有的实验动物,尚无其芳香化酶的有关资料,其性别分化机制亦不清楚.本研究采用RT-PCR的方法以简并引物扩增了稀有鮈鲫脑型芳香化酶基因Cyp19b的部分序列,其长度为1070 bp,编码357个氨基酸残基,具有典型的芳香化酶结构域.RT-PCR分析发现该基因主要在稀有鮈鲫的脑中表达,在性腺、肠、肾中也有表达;其在雌、雄脑中的表达差异不显著.在胚胎发育阶段,Cyp19b的表达从囊胚期开始,至神经胚期达到较高水平,随后下降,孵化期又有所增强,孵化10d后维持在高水平.这些结果说明Cyp19b以脑中表达为主,可能在稀有鮈鲫神经系统发育和脑的性别分化中有重要作用.