芳香化酶(P450arom)mRNA在黄鳝脑组织中的表达

为了揭示芳香化酶与黄鳝性逆转之间的内在联系和作用机制,本研究以不同性别黄鳝的脑组织为试验材料,采用实时定量PCR方法揭示P450arom mRNA在不同性别黄鳝的脑组织上的表达规律。实时定量PCR结果表明P450arom mRNA在雌性、雌雄间性、雄性黄鳝的脑组织中均有表达,其表达水平不断下降。总之,黄鳝脑组织中的芳香化酶可能参与黄鳝的性逆转过程,且与其性别转化密切相关,推测黄鳝脑组织中的芳香化酶的表达水平高低对黄鳝的性逆转可能起着决定性的作用。     

Molecular mechanism of P450c17‐II (17, 20‐lyase) regulating gonad development in female Cynoglossus semilaevis

Cytochrome P450c17 (CYP17, 17α‐hydroxylase/17,20‐lyase) is a critical enzyme in the production of androgens and estrogens in vertebrates. A 2102 bp full‐length cDNA of P450c17‐II (CYP17A2) has been isolated from the ovary of half‐smooth tongue sole, Cynoglossus semilaevis which encodes 524 amino acids. The putative P450c17‐II enzyme shares higher sequence identity with those of teleosts than with P450c17‐I of vertebrate. The similarity between the two types of tongue sole P450c17 was 48%.Semi‐quantitative RT‐PCR analysis of spatial expression showed the enzyme was specifically expressed in the ovary and the head kidney. However, temporal expression shows that P450c17‐II can be found in the brain. Furthermore, temporal expression pattern of P450c17‐II in ovary and brain revealed developmental stage‐dependency, and ovary P450c17‐II expressed remarkably throughout the whole reproductive cycle. Otherwise, the expression pattern of P450c17‐II in head kidney indicated negative ovary development‐dependence. In addition, combined with our data on P450c17‐I, T and E₂ levels, the results further endorse the critical role of P450c17‐II during shift in steroidogenesis, suggesting that P450c17‐I and ‐II may act together to this physiological process. Based on the present study, we indicate an important role for P450c17‐II during ovarian development.     

Molecular cloning, characterization expression of P450c17-I and P450c17-II and their functions analysis during the reproductive cycle in males of barfin flounder (Verasper moseri)

P450c17, a key steroidogenic enzyme, plays important roles in the production of sex steroid and cortisol. In teleost, there are two types of P450c17, P450c17-I possessing 17α-hydroxylase and 17, 20-lyase activities, and P450c17-II only possessing 17α-hydroxylase activity. This work describes the molecular cloning of the cDNA encoding the barfin flounder (Verasper moseri) P450c17-I and P450c17-II by means of RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) analyses and mRNA expression profiles analyzing by semiquantitative RT-PCR. Respectively, P450c17-I and P450c17-II mRNA levels in the testes correlated with serum testosterone (T) level, as well as gonadosomatic index (GSI) of males during specific stages of spermatogenesis. P450c17-I and P450c17-II mRNA were expressed in the testis and ovary, suggesting that both of them participate in the production of sex steroid in barfin flounder gonads. P450c17-I mRNA was undetectable; in contrast, P450c17-II mRNA was detected at the highest level in the head kidney, meaning that only P450c17-II is involved in the production of cortisol in barfin flounder. The results demonstrated that both of P450c17-I and P450c17-II participate in the production of sex steroid in male barfin flounder gonads.     

黄鳝脑芳香化酶基因cDNA的克隆及组织表达特异性分析

根据NCBI数据库报道的黄鳝芳香化酶基因的gDNA序列,设计了一对特异性引物.用Trizol试剂盒提取黄鳝脑总RNA,反转录获得cDNA第一条链,进行PCR扩增.扩增产物经过回收、连接、测序后得到了一条长1514 bp的芳香化酶基因cDNA序列.同源性比较表明该基因属于脑型P450aromB,与其他鱼类脑型P450aromB的同源性较高(>70%),与性腺型P450aromA的同源性较低(<60%),与自身性腺型芳香化酶同源性为59.5%.采用RT-PCR的方法对该基因在雄性黄鳝的各组织表达情况进行了分析,结果表明,该基因的转录本较高的表达于脑和精巢中,较低的在皮肤中表达,而在肝脏、心脏、小肠、肌肉等组织中没有检测到该基因的表达.     

仿刺参性别相关基因P450c17的克隆与序列分析

本研究克隆了仿刺参(Apostichopus japonicus)P450c17基因的全长cDNA,结果表明其cDNA的5′端UTR为256 bp,3′端UTR为250 bp,ORF为1 530 bp。在3′端有特殊序列ATTTA、终止密码子TAA和polyA加尾。其全长cDNA编码509个氨基酸的前体蛋白,预测蛋白分子量57.6 kD,理论等电点5.5。P450c17的氨基酸序列存在长度为25个氨基酸的信号肽,1个长度为22个氨基酸的跨膜区,1个糖基结合位点NHS,1个P450特有的结构域,其氨基酸序列具有明显的细胞色素P450基因家族的特征。结构域中亚铁血红素结合域中的精氨酸R和发挥活性所必须的半胱氨酸C都是保守的。这些结构组成了血红素结合环、质子传递槽和K螺旋及绝对保守EXXR基序。蛋白前体二级结构中无规则卷曲占41.22%,延伸链占12.77%,α–螺旋占46.01%,不含–折叠。氨基酸序列与其他物种氨基酸序列的同源性在35%～41%之间。系统进化树表明仿刺参与长牡蛎(Crassostrea gigas)及文昌鱼(Branchiostoma belcheri)聚为一支,黄颡鱼(Pelteobagrus fulvidraco)、鲇(Clarias gariepinus)、日本鳗鲡(Anguillajaponica)及白斑角鲨(Squalus acanthias)聚为一支,三刺鱼(Gasterosteus aculeatus)、金眼门齿鲷(Stenotomus chrysops)、斑马鱼(Danio rerio)、鲤(Cyprinus carpio)、虹鳟(Oncorhynchus mykiss)、牙鲆及青鳉(Fundulus heteroclitus)聚为一支。研究结果为进一步探讨仿刺参P450c17在性激素调控性别分化方面的功能提供参考。     

黄颡鱼脑P-450芳香化酶基因的克隆和组织表达

P-450芳香化酶（P450arom）是催化雄激素生物合成雌激素的关键酶。本文采用RT-PCR和RACE法,首次分离和克隆了黄颡鱼脑P450芳香酶基因HP450aromB,并使用荧光实时定量RT-PCR对其组织表达进行了研究。结果表明HP450aromB cDNA全长2080bp（不包括poly（A））,5′端非翻译区有25bp,3′端555bp（不包含poly（A））,阅读框（open reading frame,ORF）1500bp,编码500个氨基酸,推测的蛋白质分子量为56kDa。同源性分析显示,HP450aromB的氨基酸序列与其它鱼脑P450arom具有70％以上的同源性,与其它鱼卵巢P450arom为60％左右同源,与人胚盘和鸡卵巢P450arom则为50％左右同源;但芳香化酶高保守区包括Ⅰ-螺旋区,芳香化酶特异保守区Ⅱ及血红系结合区Ⅲ和其它鱼芳香化酶相比同源性分别为83％～96％,78％～86％和85％～100％。系统发育分析表明HP450aromB与鱼类脑P450arom属于同一分支的,并且也显示了黄颡鱼和鲶鱼分类地位最近,这和传统的分类一致。实时定量RT-PCR研究显示,HP460arom B在前脑,下丘脑,垂体、卵巢、精巢均有表达,在肝脏没表达,表达量脑部高于性腺,但雌雄鱼脑部HP450aromB的表达总量没显著差异。     

黄鳝性腺发育的研究概况

黄鳝肉质鲜美 ,营养丰富 ,具有多种药用价值 ,在国内外市场很受欢迎。长期以来 ,由于人们过量捕捉野生黄鳝以及其水域环境受到污染 ,其自然资源已日益下降 ,远不能满足市场需要和出口创汇的要求。为此 ,黄鳝人工繁殖研究迫在眉睫 ,而性腺发育是鱼类繁殖的基础。早年 ,刘建康等[     

Effects of water temperature on the gonadal development and expression of steroidogenic enzymes in the gonad of juvenile red seabream, Pagrus major

The effects of water temperature on the development of hermaphroditic gonads in red seabream (Pagrus major) and on mRNA expression of cytochrome P450 aromatase (P450arom) and 11β-hydroxylase were examined. High water temperature suppressed both expression of P450arom and 11β-hydroxylase and the development of oocytes in ovarian portion of hermaphroditic gonads.     

鳗鲡繁殖生物学研究进展

鳗鲡是优质养殖鱼类,但鳗鲡苗种完全依靠天然捕捞。近年来鳗鲡苗种的短缺限制了鳗鲡养殖业的进一步发展,开展鳗鲡人工繁殖技术研究势在必行。文章从鳗鲡的催熟与催产、产后鳗鲡的某些繁殖生物学以及鳗鲡胚胎和仔鱼发育等方面简要回顾了鳗鲡繁殖生物学的研究概况,并着重介绍了日本的最新研究进展,日本已将人工苗培育至20多厘米的成鱼,实现了实验室内鳗鲡由卵到成鱼的全人工养殖。文章最后指出人工育苗中存在的问题,并提出今后应从内因和外因两方面研究,才能真正实现鳗鲡苗种规模化生产。     

黄鳝性腺抑制差减文库的构建和分析

应用抑制性差减杂交技术构建了黄鳝(Monopterus albus)Ⅳ期卵巢和间期性腺的差减cDNA文库.正向差减杂交以间期性腺为试验方,Ⅳ期卵巢为驱动方;反向差减杂交以Ⅳ期卵巢为试验方,间期性腺为驱动方.将获得的差减cDNA片段连接质粒载体,然后转化大肠杆菌DH5α,最后获得的正、反向差减文库分别含461、678个重组子.经PCR扩增鉴定插入片段范嗣为200～2000 bp.随机选取正、反文库共100个阳性克隆测序分析,得到90个有效基因片段,与GenBank进行BLASTx和BLASTn同源比对.进一步从文库中选取2个基因用于半定量RT-PCR,对文库质量进行验证.结果表明,所建文库能够达到富集这两期性腺差异表达基因的目的.黄鳝性腺差减文库的构建,为进一步分离、鉴定黄鳝性腺发育和性逆转的相关基因奠定了基础.     

日本鳗鲡人工育苗及仔鱼饲料研究

日本通过40年对鳗鲡成熟控制技术的研究,特别是近几年来采用17,20β-dihydroxy-4-pregnen-3-one(DHP)的鳗鲡雌鱼成熟诱导技术,使高质量鳗鲡受精卵的取得具有相当高的把握性。日本水产厅养殖研究所采用以鲨鱼卵冷冻干燥粉末为主要成份并含有磷虾或其它鲜鱼虾抽提液的糊状饲料,喂食人工孵化的日本鳗鲡仔鱼取得举世嘱目的突破性进展,但柳叶状仔鱼全长达到30mm以后几乎不再摄食,出现生长停滞,并已变得十分衰弱。全面攻克日本鳗鲡人工育苗难题尚待时日,特别是研制能有效促进后期柳叶状仔鱼生长并变态的全价育苗饲料已成为目前关注的焦点。     

Partial characterization and ontogenetic development of pancreatic digestive enzymes in Japanese eel Anguilla japonica larvae

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.     

黄颡鱼卵巢P-450arom基因的克隆及组织表达

P-450芳香化酶（P450arom）是催化雄激素生物合成雌激素的关键酶。本研究采用RT-PCR和RACE（Rapid Amplification of cDNA Ends）法，分离和克隆了黄颡鱼（Pelteobasrus fulvidraco）卵巢P450芳香化酶基因HP450arom A，并使用荧光实时定量RT—PCR对其组织表达进行了分析。结果表明，HP450arom A cDNA全长1914bp[不包括poly（A）]，5’端非翻译区有13bp，3’端362bp[不包含poly（A）]，阅读框（Open Reading Frame，ORF）1539bp，翻译成513个氨基酸，计算的蛋白质分子量为58．7kD。同源性分析显示，HP450aromA的氨基酸序列与其他鱼卵巢P450arom具有60％～90％的同源性，与其他鱼脑P450arom为57％-60％同源，与鸡卵巢和人胚盘P450arom则为51％和52％同源；但芳香化酶高保守区包括Ⅰ-螺旋区，芳香化酶特异保守区Ⅱ和血红素结合区Ⅲ和其他鱼芳香化酶相比同源性分别高达68％～97％、78％-91％和71％～100％。系统发育分析表明，HP450arom A与鱼类卵巢P450arom属于同一分支，黄颡鱼和鲇鱼亲缘关系最近，这和传统的分类方法结论一致。荧光实时定量RT-PCR研究结果显示，HP450arom A在前脑、下丘脑、脑垂体、精巢、肝脏中不表达，只在卵巢中表达。这说明HP450arom A的表达具有组织特异性，并可推测其对卵巢发育起着重要作用。与本室分离的HP450arom B在卵巢的表达量相比，卵巢中HP450arom A的表达量是HP450arom B的18．7倍。     

11-ketotestosterone potentiates estrogen-induced vitellogenin production in liver of Japanese eel (Anguilla japonica)

Isolated hepatocytes from male Japanese eel at various stages of human chorionic gonadotropin (HCG)-induced spermatogenesis were cultured in the presence of estradiol-17β (E2). Hepatic vitellogenin (VTG) production in response to E2 increased during testicular development. This indicates that VTG production is influenced by gonadal maturity not only in females but also in males, where serum levels of 11-ketotestosterone (11KT) increase during testicular development. Recently, it has been confirmed that significant levels of 11KT are detected in maturing female eel. Therefore, in a next experiment, effect of 11KT on E2-induced VTG production was examined in vitro. As a result, in both male and female hepatocytes, 11KT enhanced E2-induced VTG production, although 11KT alone failed to induce VTG production.     

日本鳗鲡早期幼苗趋光性及视觉发育特征

研究了出膜后第1～9 d的日本鳗鲡(Anguilla japonica)早期幼苗,在不同光强度下趋光性分布及视觉发育组织学特征。结果显示:在8900 lux的强光条件下,日本鳗鲡早期幼苗随着发育,表现出明显的负趋光性。到9 d时,占存活幼苗52.2%的幼苗都集中在最暗的110 lux区域;而在2300 lux的弱光条件下,表现出趋弱光性。到9 d时,占存活幼苗44.6%的幼苗都集中在25 lux区域。视网膜发育的组织学观察发现,日本鳗鲡幼苗的视网膜发育首先出现的是外界膜和内界膜,最后出现的是外丛层。幼苗的整个视网膜结构到9 d时,发育为9亚层结构。视网膜中最发达的是外核层,由3种细胞构成。发育最快的是内核层,由2种细胞构成。结果表明,与日本鳗鲡早期幼苗趋光性有关的因素包括视网膜分层、视网膜厚度、内核层细胞数等多种因素。另外,本研究还探讨了日本鳗鲡幼苗在自然界可能生存的水深和光环境。