GnRH及其受体在2种真骨鱼消化系统中存在的免疫细胞化学证据

用链霉亲和素—生物素化过氧化物酶复合物(strept avidin biotin—peroxidase complex，SABC)免疫细胞化学方法，使用促性腺激素释放激素(gonadotropin—releasing hormone，GnRH)，促性腺激素释放激素受体(gonadotropinreleasing hormone receptor，GnRHR)2种抗血清对黄颡鱼(Pelteobagrus fulvidraco)和鲇(Silurus asotus)的食道、贲门、胃底、幽门、前肠、中肠、后肠和胰中的免疫活性内分泌细胞进行了定位。结果表明：在鲇的食道、胃、肠、肠固有膜、肠肌间神经丛、胰腺和胰岛中均存在着GnRH和GnRHR免疫活性阳性反应；黄颡鱼消化系统中除了在食道和胰岛中未见GnRH和GnRHR的免疫活性阳性反应外，其他部位均有阳性反应，而且GnRH和GnRHR分泌细胞的分布模式相类似。说明胃肠道中GnRH分泌细胞可能以自分泌或旁分泌方式参与消化功能的调节。本研究首次证实在鱼类的消化系统中存在着GnRH及其受体的免疫活性内分泌细胞，可为GnRH功能的多样性等研究领域提供新的形态学依据。     

中华鳖GnRH 1基因cDNA克隆及表达特征

为了解GnRH基因在中华鳖(Pelodiscus sinensis)性腺和胚胎发育过程中的表达特征,采用cDNA末端快速扩增(RACE)技术从中华鳖全脑中获得与生长生殖调控密切相关的GnRH1基因全长cDNA,并运用实时荧光定量PCR(qRT-PCR)技术检测GnRH1在成鳖不同组织和胚胎发育时期的表达水平。结果显示:中华鳖GnRH1基因cDNA全长546 bp,其中5′非编码区(5′UTR)99 bp,3′非编码区(3′UTR)168 bp,开放阅读框(ORF)279 bp,编码92个氨基酸,分子质量为10.23 ku,理论等电点pI为5.65,具有N端信号肽(1~23 aa)、核心十肽区域(24~33 aa)、断裂位点GKR(34~36 aa)及相关肽区域(37~92 aa),符合GnRH蛋白典型结构特征。系统进化树结果显示,中华鳖GnRH1基因和绿海龟(Chelonia mydas)、墨西哥箱龟(Terrapene carolina mexicana)及西部锦龟(Chrysemys picta bellii)GnRH1基因聚为一支。qRT-PCR结果表明,GnRH1基因在中华鳖雌雄个体的8个组织中均有表达,在脑和性腺组织中高表达,且具有性别差异,雄性中华鳖中的表达显著高于雌性(P<0.05);在10个胚胎发育时期均表达,且随发育时间的后移,表达量显著增加,在第16期达到峰值。GnRH1基因可能在中华鳖生长及性腺分化中具有重要作用。     

介绍效果更好的鱼类催产合剂——高效鱼类催产合剂2号和3号

鱼类促性腺激素(GtH)的分泌和生殖活动已经证明为双重的神经内分泌系统所调节，即促性腺激素释放激素(GnRH)刺激GtH分泌和多巴胺作为促性腺激素释放的抑制因素(GRIF)抑制GnRH的作用以及GtH的自行释放。我们先前对养殖鱼类的研究已证明多巴胺受体的拮抗物(如pimozide，PIM)，排除剂(如reserpine，RES)和多巴胺合成抑制剂都能显著增强LRH-A诱导GtH分泌和排卵的作用。     

半滑舌鳎促性腺激素α亚基cDNA的克隆及组织表达特征

采用同源克隆和末端快速扩增（RACE）方法,从半滑舌鳎脑垂体中克隆了促性腺激素α亚基（CGα）全长cDNA（GenBank序列登录号：JQ364953）.半滑舌鳎CGα基因全长685bp,其开放阅读框384bp,编码含127个氨基酸的蛋白,N端33个氨基酸为信号肽.半滑舌鳎CGα成熟肽与其他脊椎动物CGα成熟肽结构特征相似,具有10个半胱氨酸残基和两个N-糖基化位点.CGα成熟肽序列分析表明,半滑舌鳎CGα与鲽形目和鲈形目鱼类CGα同源性为60％～70％,与鲤形目鱼类CGα同源性为55％～60％.实时荧光定量RT-PCR结果表明,半滑舌鳎CGα mRNA在被检测的12个组织中均有表达,除头肾和脾脏、脾脏和肝脏间表达量差异不显著外,其他组织间表达量差异显著（P＜0.05）;CGα mRNA在垂体组织中大量表达,其次是鳃、肾脏、肌肉、卵巢和脑组织,而在心脏、头肾、肝和脾等组织中表达量很低.本研究为进一步探讨促性腺激素在半滑舌鳎繁殖生理中的作用机制奠定基础.     

半滑舌鳎(Cynoglossus semilaevis) gnrh2基因克隆、组织分布及卵巢成熟过程中表达分析

为了研究下丘脑神经肽促性腺激素释放激素(Gonadotropin-releasing hormone 2,GnRH2)在半滑舌鳎(Cynoglossus semilaevis)卵巢成熟过程中的生理作用,本研究通过RT-PCR及RACE方法获得了半滑舌鳎GnRH2全长cDNA序列;通过实时荧光定量PCR(qPCR)对gnrh2 mRNA的组织分布以及卵巢成熟过程中的时空表达特性进行了分析.结果显示,半滑舌鳎GnRH2全长cDNA序列为538 bp(不包括polyA尾),其中,5'非编码区(Untranslated region,UTR)为154 bp,3'UTR为126 bp,开放阅读框(Open reading frame,ORF)为258 bp,编码85个氨基酸的前体多肽,其分子量及等电点分别为9.69 kDa和8.55.GnRH2前体多肽由信号肽、GnRH2十肽、酶切位点(GKR)以及GnRH相关肽共4部分组成.序列比对分析发现,GnRH2在鱼类中同源性极高,尤其是十肽(QHWSHGWYPG)在所有硬骨鱼类中完全相同.半滑舌鳎GnRH2与鲈形目同源性最高(89.41％-90.5 9％),其次为鲽形目、鲑形目和鲍形目(78.82％-85.88％),与鲤形目同源性最低(61.18％-71.76％).gnrh2 mRNA主要在脑中表达,在垂体及其他外周组织中表达量极低.此外,组织学分析显示,半滑舌鳎卵巢发育共分为5个时期(Ⅱ、Ⅲ、Ⅳ、Ⅴ和Ⅵ期).在卵巢成熟过程中,脑gnrh2 mRNA表达量在卵黄生成期(Ⅲ期)显著性增加,达到峰值;随后表达量急剧下降,在成熟期(Ⅴ期)达到最小值;在排卵后期(Ⅵ期)又显著性增加.然而,在卵巢成熟过程中,垂体gnrh2 mRNA表达量在卵黄生成后期(Ⅳ期)显著性降低,随后在成熟期(Ⅴ期)有所增加,但在排卵后期(Ⅵ期)又急剧下降.上述研究结果表明,脑GnRH2可能参与了半滑舌鳎卵巢发育过程.     

牙鲆蛋白质感染因子蛋白基因的克隆和结构分析

采用RT-PCR方法分离牙鲆（Paralichthys olivaceus）蛋白质感染因子蛋白编码基因序列。推测的牙鲆蛋白质感染因子蛋白由472个氨基酸组成，分子量约49．6kD，具有信号肽、短肽顺接重复、疏水区、糖基化位点、二硫键、糖基缩醛磷肌醇锚定位点等蛋白质感染因子蛋白特征结构域，与红鳍东方纯（Fugu Rubripes）和大西洋鲑（Atlantic salmon）蛋白质感染因子蛋白的相似性分别为71．9％和66．4％。牙鲆蛋白质感染因子蛋白编码基因序列的获得为蛋白质感染因子进化与功能研究以及可能存在的鱼类传染性海绵样脑病研究奠定了基础。     

牙鲆肌肉生长抑素(MSTN)基因克隆

采用同源克隆及基因组步移的方法，分离克隆了牙鲆肌肉生长抑素（MSTN）基因。经过序列分析及cDNA验证，牙鲆MSTN基因具有3个外显子和2个内含子，编码377个氨基酸。5`侧翼区含有8个TATA框，一个CAAT框，6个E框；3`侧翼区含有加尾信号。通过同源分析，牙鲆MSTN C末端含有9个保守半胱氨酸残基和一个RVRR蛋白酶酶切位点；通过进化树分析，牙鲆MSTN与鱼类MSTN基因聚为一支。RT-PCR分析表明，牙鲆MSTN在胚胎发育中不表达或表达量较低，说明MSTN在牙鲆胚胎发育中并不起重要作用；其在各组织中的表达，随个体和环境的不同而有差异，暗示MSTN的表达受外界因素调控。     

斜带石斑鱼抗菌肽hepcidin基因克隆及其成熟肽的原核融合表达

文章根据已报道的硬骨鱼类hepcidin cDNA序列设计简并引物，通过RT—PCR从重要海水经济鱼类斜带石斑鱼（Epinephelus coioides）肝脏中扩增出1条具有完整开放阅读框的246bpcDNA序列，Blast分析表明该序列是鱼类hepcidin基因家族的一员，被命名为斜带石斑鱼hepcidin样抗菌肽前体，该cDNA所推导的氨基酸序列有如下特点：1）信号肽序列与多数鱼类hepcidin信号肽的同源性在67％～87％之间；2）C端20个氨基酸序列具有绝大多数动物hepcidin成熟肽的共同典型保守序列特征，即在对应位置具有8个Cys；3）序列中不具有已报道的hepcidin前体肽转化酶作用典型基序[RX（K／R）R]；4）与GenBank中已注册的2条斜带石斑鱼hepcidincDNA序列（GU391241和GU391242）所推导的氨基酸序列同源性分别为36％和33％，且NJ进化树显示不与这2条序列聚为一枝，表明该序列是斜带石斑鱼hepcidin基因家族中一种新亚型，其预测成熟肽融合表达载体成功在大肠杆菌（Escherichiacoli）中表达，为后续研究奠定基础。     

尼罗罗非鱼白细胞介素21基因 (OnIL-21)及其受体基因 (OnIL-21R)克隆及表达分析

白细胞介素21（interleukin 21,IL-21）是一种多效性细胞因子,主要通过其专一性受体（IL-21R）行使免疫调节功能。为研究IL-21及其受体IL-21R在鱼类抵御病原菌的免疫应答过程中的作用,本研究利用反转录PCR（RT-PCR）技术成功克隆了尼罗罗非鱼IL-21基因（OnIL-21）及其受体IL-21R基因（OnIL-21R）,对其进行生物信息学分析,并利用实时荧光定量PCR（qRT-PCR）技术检测其mRNA水平的表达。结果显示,OnIL-21及其受体OnIL-21R的开放阅读框为420 bp和1 548 bp,分别编码139和515个氨基酸。经氨基酸序列预测分析,OnIL-21为分泌型蛋白,具有一个Ig-like结构域;而OnIL-21R是跨膜蛋白,具有典型的γ-c链,2个基因的蛋白序列都具有多个保守的磷酸化位点和糖基化位点。系统进化树结果表明,OnIL-21和OnIL-21R均与斑马拟丽鱼IL-21和IL-21R的亲缘关系最近。组织差异性表达分析发现,OnIL-21在皮肤和心脏组织中表达量较高,在肌肉中较低;而OnIL-21R在鳃和脾脏组织中表达量最高,在肌肉中最低。无乳链球菌和嗜水气单胞菌体内感染和体外刺激白细胞后,头肾和脾脏中OnIL-21和OnIL-21R表达量均呈现显著上调,说明OnIL-21和OnIL-21R可能参与尼罗罗非鱼病原菌感染的免疫应答过程。此外,重组蛋白(r)OnIL-21刺激脾脏白细胞后,OnIL-21R和炎症相关因子（IL-1β、TNF-α、IFN-γ、IL-6和IL-10）的mRNA表达均发生显著上调,表明OnIL-21可调控其受体基因OnIL-21R的表达及参与调控机体的炎症反应。上述研究结果阐述了尼罗罗非鱼IL-21与其受体IL-21R的分子生物学特征,并初步阐明了二者参与了机体病原菌感染的免疫应答过程,为进一步探究IL-21通过其受体IL-21R调控机体免疫反应的作用机制和信号通路提供了重要参考。     

鳜hepcidin cDNA的分子克隆及序列分析

Hepcidin是近年来发现的一类具有独特性质的抗菌肽，根据C-enBank中收录的鱼类抗菌肽hepcidin的cDNA序列设计一对简并引物，用RT-PCR方法从鳜（Sinipercachuatsi）肝脏组织中克隆到hepcidin的cDNA，并构建pMD18-T-hep-cidin载体进行序列测定和分析。结果表明，该cDNA长为381bp，其中第20-277位是该基因的开放阅读框（ORF），编码86个氨基酸，形成由信号肽（24个残基）、前肽（42个残基）和成熟肽（20个残基）3部分序列组成的前体肽，与已报道的其他鲈形目鱼类hepcidin相比较，核苷酸序列的同源性为72．2％-92．0％，所推导的成熟肽与包括人类在内的其他生物hepcidin成熟肽的同源性在50％-86．4％，都含有8个保守的cys残基，可形成4个链内二硫键。鳜hepcidin cDNA片段的获得为以后的重组表达奠定了实验基础，也为抗菌肽hepcidin家族找到了新的成员。     

Effect of dietary supplementation with taurine, β-alanine andGABA on the growth of juvenile and fingerling Japanese flounder Paralichthysolivaceus

ABSTRACT:   Three diets supplemented with taurine, β-alanine andGABA and a control diet were fed to juvenile and fingerling Japaneseflounder to investigate the effects of the diets on growth and metabolicchanges of free amino acids in whole body and tissues. In experimentI, three diets supplemented with 1% each of taurine, β-alanineand GABA and a control diet were fed to juvenile Japanese flounderwith an initial mean body weight of 0.4 g for 4 weeks at20°C. In experiment II, the taurine-supplemented diet anda control diet were fed to fingerling Japanese flounder with an initialmean body weight of 15 g for 4 weeks at 20°C.Only supplementation of taurine in the diet of juvenile flounderimproved their growth performance in experiment I, but fingerlinggrowth performance of experiment II was not significantly relatedto taurine supplementation in the experimental diet. These resultssuggest that there is a greater requirement for taurine for thegrowth of juvenile Japanese flounder than fingerling Japanese flounder.     

Characterization of the receptor for gonadotropin-releasing hormone in the pituitary of the African catfish,Clarias gariepinus

Receptors for gonadotropin-releasing hormone (GnRH) were characterized using a radioligand prepared from a superactive analog of salmon GnRH (sGnRH), D-Arg⁶-Pro⁹-sGnRH-NEt (sGnRHa). Binding of¹²⁵I-sGnRHa to catfish pituitary membrane fractions reached equilibrium after 2 h incubation at 4°C. Displacement experiments with several GnRH analogs as well as other peptides, demonstrated the specificity of¹²⁵I-sGnRHa binding. Specific binding was enhanced in the presence of the cation chelator ethylene bis (oxyethylenenitrilo) tetra-acetic acid (EGTA), indicating an inhibitory effect of cations on GnRH-receptor binding. The binding of¹²⁵I-sGnRHa to pituitary membranes was found to be saturable at radioligand concentrations of 5 nM and above. A Scatchard analysis of the saturation data suggested the presence of a single class of high-affinity binding sites (Ka=0.901±0.06×10⁹M^–1, Bmax=1678±150 fmol/mg protein). A comparative study on¹²⁵I-sGnRHa binding to pituitary membrane fractions of male and female catfish, indicated that there were no differences in binding affinity and binding capacity between both sexes. The results demonstrate the presence of specific, saturable GnRH receptors in the African catfish pituitary.     

红螯螯虾冷休克蛋白Y-box编码基因的克隆及表达分析

采用RT-PCR和RACE技术,从红螯螯虾(Cherax quadricarinatus)血细胞中分离到冷休克蛋白Y-box编码基因的cDNA序列。结果显示,冷休克蛋白Y-box编码基因的长度为1 733 bp,其中包括82 bp的5'非翻译区、676 bp的3'非翻译区和975 bp的编码序列,可编码324个氨基酸。理论相对分子质量和等电点分别为35 800和9.81。结构域分析和序列比对显示,红螯螯虾冷休克Y-box蛋白由3部分组成:富含丙氨酸或脯氨酸区域、冷休克结构域和带电荷区域,其中冷休克结构域高度保守。系统进化树分析表明,红螯螯虾冷休克Y-box蛋白与水蚤等节肢动物冷休克Y-box蛋白聚类,且三级结构非常相似。最适温度养殖条件下,冷休克蛋白Y-box的虾组织分布特异性研究表明,红螯螯虾神经组织的冷休克蛋白Y-box转录活性最强,而后依次为肝脏、血淋巴和鳃,在肠和心脏中也有活性,在肌肉组织中转录保持较低水平,揭示神经组织是重要的冷休克Y-box蛋白表达区。     

mRNA expression of GnRH variants and receptors in the brain,pituitary and ovaries of pejerrey (<Emphasis Type="Italic">Odontesthes bonariensis</Emphasis>) in relation to the reproductive status

The present study examined the differential mRNA expression levels of three forms of GnRH (sGnRH, pjGnRH and cGnRH-II) and two forms of GnRH receptor (pjGnRH-R I and pjGnRH-R II) in the brain, pituitary, and ovaries of pejerrey in relation to the reproductive status. The analysis revealed the presence of significant amounts of mRNA of the three GnRH forms while the ovaries showed only two (sGnRH and pjGnRH). The GnRH receptor II was found ubiquitously in the brain, pituitary, and ovaries while the form I was detected only in the brain. The levels of pjGnRH mRNA in the brain and pjGnRH-R II in the pituitary gland varied in correlation with the ovarian condition. However, brain sGnRH and pjGnRH-R I mRNA levels reached a maximum during early stages of ovarian development. In contrast, the brain levels of cGnRH-II mRNA showed no variation. The present study also shows a good correlation of ovarian sGnRH and pjGnRH-R II mRNA levels with the reproductive condition, suggesting that these molecules are may be involved in the regulation of pejerrey ovarian function.     

Expression of taurine transporter in response to hypo-osmotic stress in the mantle of Mediterranean blue mussel

Expression of taurine transporter in response to osmotic stress was investigated at the protein level in the mantle of the Mediterranean blue mussel by using the specific antibody raised against the carboxy-terminal region of the deduced amino acid sequence of mussel taurine transporter. Immunohistochemical observation revealed that taurine transporter was expressed in the mantle and the expression was up-regulated in response to hypo-osmotic stress, while down-regulated in response to hyper-osmotic stress. Western blot analysis revealed major protein bands corresponding to 62 kDa and 65 kDa. In response to hypo-osmotic stress, the 62 kDa band became more intense, while it became less intense when the ambient osmolality was elevated. These results suggested that the 62 kDa taurine transporter would be implicated in hypo-osmotic adaptation.     

Influence of dietary protein on digestive enzyme activity, growth and tail muscle composition in redclaw crayfish, Cherax quadricarinatus (von Martens)

This study was conducted to evaluate the effects of dietary protein on digestive enzyme profiles, growth and tail muscle composition in the freshwater redclaw crayfish, Cherax quadricarinatus. Crayfish were fed five diets that consisted of a commercial crayfish pellet and experimental diets containing 13%, 18%, 25% or 32% crude protein (CP), for a period of 12 weeks. Analysis of digestive enzyme profiles from the midgut gland (MG) revealed a positive correlation between protease, amylase and cellulase activities and dietary protein level. For all treatments, carbohydrase activity levels (cellulase and amylase) were significantly higher than those detected for protease. As dietary protein was elevated, there was a general increase in specific growth rate (SGR), with the highest SGR (0.58 ± 0.06) values observed in crayfish fed the diet containing 25% CP. Feed conversion ratio (FCR) ranged between 5.84 and 6.97 and did not differ significantly among the treatment groups including the reference diet, with the exception of the low‐protein diet (13% CP) which showed an FCR of 9.31. Finally, regression analysis revealed a strong positive correlation between the level of dietary protein and CP content in the tail muscle (P=0.004; r²=0.99).     

Distinct expression of GnRH genes in the red seabream brain

This paper reports the molecular cloning of a cDNA encoding the precursor of seabream gonadotropin-releasing hormone (prepro-sbGnRH) and the localization of salmon GnRH (sGnRH) and seabream GnRH (sbGnRH) expressing neurons in the brain of the red seabream (Pagrus major). The cloned prepro-sbGnRH cDNA has a 285 bps open reading frame encoding a 23 amino acid signal peptide, a 10 amino acid sbGnRH, the cleavage site (Gly-Lys-Arg), and a 59 amino acid GnRH-associated peptide. The expression of sGnRH and sbGnRH peptides, and prepro-sGnRH and prepro-sbGnRH mRNA were studied using immunocytochemistry and non-radioactive in situ hybridization, respectively. We found cell bodies that reacted positively with both the sGnRH cRNA probe and anti-sGnRH serum, but not with the sbGnRH cRNA probe or anti-sbGnRH serum in the ganglion of the terminal nerve. Cell bodies that reacted positively with the sbGnRH cRNA probe, anti-sbGnRH serum, and anti-sGnRH serum, but negatively with the sGnRH cRNA probe were found in the preoptic area (POA). Immunocytochemistry showed that a distinct bundle of axons arises in the POA which projected to the pituitary gland. These results suggest that sbGnRH is the most relevant hypophysiotropic form of GnRH.     

Effect of dietary protein and lipid source on the growth, survival, condition indices, and body composition of marron, Cherax tenuimanus (Smith)

A trial was conducted in 12 purpose-built, commercial, drainable, earthen ponds to evaluate the effect of fish and plant protein and lipid source on the growth, condition indices, and body composition of marron (Cherax tenuimanus). Juvenile marron (1.3±0.28 S.E. g) at the stocking densities of three per square meter were fed for a period of 1 year with four different formulated isoenergetic practical diets (D₁, D₂, D₃, and D₄). Three of the test diets (D₁, D₂, and D₃) were isonitrogenous whereas the fourth test diet (D₄) was protein-free. Protein and lipid sources in D₁ were from Lupin (Lupinus albus) whereas protein and lipid sources in D₂ and D₃ were from fish meal. Fish oil (3.5%) was added to D₁, D₃, and D₄ whereas sunflower oil was added to D₂ and D₄ in order to make them isoenergetic. The four test diets were randomly allocated to three replicate ponds.

The lack of protein in D₄ did not significantly influence (P>0.05) the mean final weight and specific growth rate of marron. Survival was low in all ponds (13.82–34.66%) but feeding with D₄ resulted in a significantly (P<0.05) higher survival than marron fed with D₁ and D₂. Feeding a diet containing a combination of fish protein and fish oil (D₃) resulted in significantly higher (P<0.05) wet tail muscles-to-body weight ratio than was observed with other diets. Tail muscles protein level of all marron was significantly lower (P<0.05) at the end of the trial than at the beginning. EPA and DHA in hepatopancreas and tail muscles of marron were affected by the four test diets. The inclusion of plant protein in formulated diets had a negative impact on the pond environment due to significantly higher unionised ammonia levels that resulted in lower survival. Juvenile marron fed with a plant protein diet had significantly lower protein levels in their hepatopancreas compared to those fed with diets containing animal protein. Feeding marron with lupin protein source (D₁) and plant oil (D₂) for 1 year did not alter the lipid content of their hepatopancreas. Four test diets had no influence on the fat content of marron hepatopancreas; however, these test diets significantly reduced the protein content of the tail muscles of marron.

Environmental variables, particularly temperature, nitrogen metabolites, and the natural productivity of the ecosystem, greatly influenced the nutritional requirements of the juvenile marron under culture.