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胡子鲇脑型芳香化酶基因全长cDNA克隆及表达
引用本文:孙晶,李广丽,朱春华,吴天利,邓思平.胡子鲇脑型芳香化酶基因全长cDNA克隆及表达[J].中国水产科学,2012,19(3):408-415.
作者姓名:孙晶  李广丽  朱春华  吴天利  邓思平
作者单位:广东海洋大学水产学院,广东湛江524025;南海水产经济动物增养殖广东普通高校重点实验室,广东海洋大学,广东湛江524025
基金项目:广西科学研究与技术开发计划重点项目
摘    要:采用RT-PCR和RACE法,克隆了胡子鲇(Clarias fuscus)脑型芳香化酶基因Cyp19a1b,应用荧光实时定量PCR检测其在前脑、下丘脑、脑垂体、肝、精巢和卵巢6种组织,以及性腺分化前后(出膜后12~30 d)全鱼中的mRNA表达。结果表明,Cyp19a1b cDNA全长2 347 bp,5′端非编码区219 bp,3′端不包括poly(A)]596 bp,开放阅读框(ORF)1 503 bp,编码500个氨基酸,推测其编码蛋白质分子量为56.388 kD。序列分析及分子系统进化树结果表明,胡子鲇Cyp19a1b氨基酸序列与非洲鲇(Clarias gariepinus)Cyp19a1b同源性最高,达95.6%;与南方大口鲇(Silurusmeridionalis)、黄颡鱼(Pelteobagrus fulvidraco)、斑马鱼(Danio rerio)、斑点叉尾(Ictalurus punctatus)、赤点石斑鱼(Epinephelus akaara)、鲤(Cyprinus carpio)、鲫(Carassius auratus)及稀有鲫(Gobiocypris rarus)Cyp19a1b同源性达75%以上,但与上述鱼类的性腺型芳香化酶基因(Cyp19a1a)同源性低于62%,表明其为脑型芳香化酶,同源性分析结果与根据传统形态学和生化特征分类的结果相一致。荧光实时定量PCR结果显示,Cyp19a1b在胡子鲇上述组织中均有表达,其中下丘脑中表达量最高,肝和精巢最低,在脑部的表达存在明显的性别差异(P<0.05)。此外,在胡子鲇性腺分化前(出膜后12 d)Cyp19a1b即开始表达,但分化前后表达量无显著性差异(P>0.05)。结果暗示Cyp19a1b可能不是引起胡子鲇性腺分化的直接因素,但其可能通过"下丘脑垂体性腺轴"对性腺分化过程产生间接影响。

关 键 词:胡子鲇  P450芳香化酶基因  RT-PCR  性腺分化  表达
修稿时间:2012/5/14 0:00:00

Molecular cloning and expression of Cyp19a1b cDNA in Clarias fuscus
SUN Jing,LI Guangli,ZHU Chunhu,WU Tianli,DENG Siping.Molecular cloning and expression of Cyp19a1b cDNA in Clarias fuscus[J].Journal of Fishery Sciences of China,2012,19(3):408-415.
Authors:SUN Jing  LI Guangli  ZHU Chunhu  WU Tianli  DENG Siping
Institution:1,2 1.Fisheries College,Guangdong Ocean University,Zhanjiang 524025,China; 2.Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal of Guangdong Higher Education Insti-tutes,Guangdong Ocean University,Zhanjiang 524025,China
Abstract:A cDNA encoding Cyp19a1b was derived from Clarius fuscus using RT-PCR and RACE.The cDNA was 2 347 bp with a 219 bp 5′UTR,a 596 bp 3′UTR(excluding poly(A)),and a 1 503 bp ORF,which encoded 500 amino acids and had a predicted mol wt of 56.388 kD.Sequence and phylogenetic analyses indicated that the C.fuscus Cyp19a1b shared 95.6% sequence identity with clarias gariepinus and >75% identity with Silurus merid-ionalis,Pelteobagrus fulvidraco,Danio rerio,Ictalurus punctatus,Cyprinus carpio,Carassius auratus,Gobio-cypris rarus,and Epinephelus akaara.In contrast,there was low sequence identity(<62%) with Cyp19a1a for these species.Therefore,the gene was classified into the Cyp19a1b subfamily.This is consistent with the classifi-cation based on traditional morphology and biochemistry.Cyp19a1b mRNA was expressed primarily in the fore-brain,hypothalamus,and pituitary,and to a lesser extent in the liver,testis and ovary.We observed differences in the level of expression in brain between males and females(P<0.05).Cyp19a1b was expressed prior to sex dif-ferentiation in C.fuscus,but there was no difference in the level of expression between prior and post to sex dif-ferentiation(12-30 d after hatching).Our results suggest that Cyp19a1b is not directly involved in mediating sex differentiation in C.fuscus,but may play an indirect role by acting on the hypothalamic-pituitary-gonadal axis.
Keywords:Clarias fuscus  Cyp19a1  RT-PCR  sex differentiation  mRNA expression
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