水生生物学报2009，Vol.33Issue(4)：748-755,8.DOI:10.3724/SP.J.0000.2009.40748
稀有鮈鲫脑芳香化酶cDNA片段的克隆与表达分析
CLONING AND EXPRESSIONAL ANALYSIS OF Cyp19b GENE FRAGMENT IN GOBIOCYPRIS RARUS
摘要
Abstract
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.关键词
稀有鮈鲫/芳香化酶/Cyp19b/基因克隆/表达模式Key words
Gobiocypris rarus/P450 aromatase/Cyp19b/Gene cloning/Expression pattern分类
生物科学引用本文复制引用
曹梦西,杨玉慧,江文波,王玉凤,胡炜,赵浩斌..稀有鮈鲫脑芳香化酶cDNA片段的克隆与表达分析[J].水生生物学报,2009,33(4):748-755,8.基金项目
国家自然科学基金(30671609) （30671609）
淡水生态与生物技术国家重点实验室开放基金(2009FB18)资助 （2009FB18）
