鲈3种视黄醇类X受体基因cDNA的克隆和组织表达

视黄醇类X受体(retinoid X receptor,RXR)属于配体激活的核受体家族,在调节细胞内各种信号途径中起重要作用.采用RT-PCR和RACE技术,分离到鲈RXR基因3种亚型RXRα、RXRβ和RXRγ的cDNA序列.结果表明,得到的RXRα是长度为1 686 bp 3′末端不完整的cDNA序列,其中包括364 bp的5′非翻译区和1 322 bp的编码序列,可编码440个氨基酸.RXRβ cDNA全长为1 741 bp,其中包括150 bp的5′非翻译区,256 bp的3 ′非翻译区和1 335 bp的开放阅读框,共编码444个氨基酸,理论分子量为49.5 ku.RXRγ cDNA全长为1 847 bp,其中5′非翻译区为88 bp,3 ′非翻译区为397 bp,开放阅读框为1 362 bp,共编码453个氨基酸,分子量约为49.9 ku.氨基酸序列分析显示,鲈RXRα、RXRβ和RXRγ和其它物种的RXRs结构类似,在DNA结合区有P box和D box结构,配体结合区包括12个α螺旋和2个β折叠结构,H12的C端含有AF-2的激活区.系统进化树分析表明,鲈RXRα、RXRβ和RXRγ分别与其他动物的相应亚型聚类.组织分布特异性研究表明,鲈RXRα在肌肉、心脏、眼、大脑、肠、肾脏、脂肪、脾脏、鳃和肝脏10种组织中均有表达,但表达量较低；RXRβ在这10组织中都有较高表达,但心脏中表达量较低；在所检测的10种组织中,RXRγ都有表达,在肌肉、肠、肾脏、脂肪和脾脏表达量较高.     

虾夷扇贝肌动蛋白基因cDNA序列克隆与分析

采用RT-PCR和RACE法从虾夷扇贝闭壳肌中分离和克隆了肌动蛋白基因cDNA全长序列.肌动蛋白基因cDNA全长1775 bp(不包括poly A),5′端非编码区94 bp,3′端非翻译区551 bp,阅读框1131 bp,编码376个氨基酸.在基因组DNA中,该基因被一个内含子分为两段,内含子位于第42和第43个氨基酸之间,长度为2041 bp.系统发育分析显示该肌动蛋白属于α类型.     

草鱼ghrelin基因的分子克隆与组织分布及其摄食调控作用分析

ghrelin是一种在脊椎动物摄食调节过程中起重要作用的脑肠肽,具有明显的摄食促进作用。实验利用同源克隆技术获得了草鱼ghrelin基因的cDNA序列和DNA序列,其中cDNA序列全长506 bp,包括90 bp的5′端非编码区(5′-untranslated region,5′UTR),312 bp的开放阅读框(open reading frame,ORF),以及104 bp的3′端非编码区(3′-untranslated region,3′UTR)。开放阅读框编码的103个氨基酸的ghrelin前体肽,经剪切加工后形成含有19个氨基酸的成熟肽。氨基酸序列分析结果显示,草鱼ghrelin与硬骨鱼类ghrelin相似度最高,而与其他脊椎动物相似度较低,同时草鱼ghrelin成熟肽N端的"活性中心"(active core)为鲤科鱼类中常见的GTSF形式。与大多数硬骨鱼类的ghrelin基因结构相同,草鱼ghrelin基因也包括4个外显子和3个内含子。荧光定量PCR检测到ghrelin mRNA大量分布于草鱼的前肠和脾,脑、肾、肝、肌肉、皮和鳔等组织也有ghrelin mRNA分布。草鱼脑和肠中的ghrelin表达水平在摄食后下降,随着饥饿时间的延长表达水平逐步升高,最后维持在较高水平,表明ghrelin作为摄食启动信号对草鱼的摄食活动起到了促进作用。     

稀有■鲫Foxo3a、Foxo3b基因克隆和表达分析

利用RACE技术自稀有■鲫的卵巢中克隆叉头蛋白O3a(Foxo3a）、叉头蛋白O3b(Foxo3b）基因的全长cDNA序列，采用RT-PCR、qRT-PCR和原位杂交法检测其组织分布、时序表达、细胞定位和雷帕霉素处理后的表达。结果显示，Foxo3a基因全长cDNA序列为2562bp，包括242bp的5′端非编码区、367bp的3′端非编码区、1953bp的开放阅读框，编码650个氨基酸；Foxo3b基因全长cDNA序列为2804bp，包括470bp的5′端非编码区、375bp的3′端非编码区、1959bp的开放阅读框，编码652个氨基酸。Foxo3a基因只在脑和卵巢中表达，在其他组织中不表达；Foxo3b基因在所检测的组织中均有表达，在脑和卵巢中的表达量较高。随着卵巢的发育，Foxo3a基因表达量呈逐渐升高的趋势，Foxo3b基因表达量未出现显著变化。原位杂交结果显示，Foxo3a、Foxo3b基因定位于卵巢Ⅱ、Ⅲ、Ⅳ时相的生殖细胞和滤泡细胞中。雷帕霉素处理影响了卵巢中Foxo3a基因的表达，对Foxo3b基因的表达无显著影响。试验结果表明，Foxo3a和Foxo3b基因可能在稀有■鲫...     

中华鲟热休克蛋白hsp30基因cDNA的克隆及其在高温胁迫下的表达分析

实验克隆了中华鲟(Acipenser sinensis)热休克蛋白hsp30基因cDNA的全长、分析了其分子结构与特征,并研究了其在高温胁迫下的表达水平。结果显示,中华鲟hsp30基因cDNA序列全长为1 037 bp,其中开放阅读框(ORF)636 bp,5′端非编码区(5′UTR)38 bp,3′端非编码区(3′UTR)363 bp,共编码211个氨基酸。氨基酸多序列比对发现含有一个保守的α晶状体结构;系统进化分析显示,中华鲟HSP30与鱼类HSP30聚为一支,与小体鲟HSP30氨基酸序列相似性最高,为79%。荧光定量PCR结果表明,中华鲟hsp30基因在皮肤中的表达量最高,肝脏次之,在肠中的表达量最低。高温胁迫后,心脏、脾脏、肾脏和皮肤中hsp30基因表达量均显著增加,表明这些器官在中华鲟应对高温胁迫中可能起着重要作用。     

鳜胃蛋白酶原基因cDNA全长的克隆与序列分析

利用RT–PCR和cDNA末端快速扩增法(RACE)克隆鳜(Siniperca chuatsi)胃蛋白酶原基因cDNA全长序列，并对该基因的结构特征和系统进化关系进行了分析。鳜胃蛋白酶原基因cDNA序列全长1367 bp，5′端非翻译区43 bp，3′端非翻译区187 bp，开放阅读框(ORF)1137 bp，共编码378个氨基酸。鳜胃蛋白酶原氨基末端存在信号肽和激活肽序列，序列中含有催化活性必需的2个天冬氨酸残基和构成二硫键的6个半胱氨酸残基。鳜胃蛋白酶原氨基酸序列与其他脊椎动物胃蛋白酶原氨基酸序列的同源性为59.9%～91.2%，表明胃蛋白酶原基因在脊椎动物的长期进化中比较保守。鳜胃蛋白酶原基因的成功克隆不仅为进一步研究该基因的时空表达奠定基础，而且为鱼类胃蛋白酶原的分子特征和进化提供了新的资料。     

斑节对虾细胞周期蛋白Y基因克隆与原核表达

为了研究细胞周期蛋白Y(cyclin Y)在斑节对虾(Penaeus monodon)卵巢发育中的作用,从斑节对虾转录组数据中筛选获得cyclin Y基因部分序列,采用SMART-RACE方法克隆得到斑节对虾细胞周期蛋白Y(Pm-cyclin Y)基因c DNA全序列。Pm-cyclin Y基因c DNA全长1576 bp,其中包含108 bp的5′非编码区(5′UTR)和439 bp的3′非编码区(3′UTR)以及1029 bp的开放阅读框(ORF),可编码342个氨基酸。生物信息学分析显示,其编码的氨基酸序列有1个保守的周期蛋白框(cyclin box)同源结构域(172~257 aa),预测的分子量约为37.6 k D,理论等电点6.64。实时定量PCR显示其m RNA在卵巢的表达量显著高于其他组织(P0.05);并且在卵巢5个不同发育期都有表达,在III期卵巢中的表达量最高。本研究通过原核表达方法对Pm-cyclin Y进行重组表达,为其蛋白质功能方面的研究奠定了基础。     

镉胁迫对黄鳝醛酮还原酶基因表达的影响

采用RACE-PCR技术获取黄鳝醛酮还原酶(Eakr)基因5′cDNA末端和3′cDNA末端，采用基因特异性引物扩增其内含子序列，获得其基因结构；将黄鳝养殖在15 mg/L CdCl₂的曝气水中，分析Cd²⁺处理对Eakr基因表达的影响；采用液体培养和斑点展示法，比较重组Eakr基因菌株和阴性对照菌株在Cd²⁺暴露下的存活率及生长活力。研究结果表明，Eakr基因全长4468 bp,包含1026 bp的开放阅读框，编码341个氨基酸，5′端非编码区74 bp, 3′端非编码区195 bp。荧光定量PCR检测发现，Eakr基因在黄鳝的脑、血细胞、心脏、肠道、肾脏、肝脏、肌肉、性腺、皮肤、脾脏、胃中均有表达，在肝脏中表达水平最高(P<0.05)。Cd²⁺胁迫下，肝脏中Eakr基因与Nrf2基因mRNA表达水平均显著上升；在各检测时间点，Eakr基因表达水平相比对照组均差异极显著(P<0.001),而Nrf2基因表达水平在处理后6、9、12 h差异极显著(P<0.001);含Eakr基因的...     

脊尾白虾不同发育期线粒体基因组甲基化特征分析

为研究不同发育阶段脊尾白虾线粒体基因组表观遗传学特征,本研究在筛选硫化测序引物的基础上,对不同发育期的脊尾白虾线粒体基因组甲基化特征进行了分析。经引物筛选验证,选取了4对引物在脊尾白虾的受精卵、幼体期、仔虾期和成虾期共4个发育期进行甲基化特征的分析,结果表明,除ND5基因3′端序列(21#引物扩增区)无甲基化现象外,COX3基因的起始区序列(13#引物扩增区)、COX3基因的3′端序列(16#引物扩增区)和ND5基因5′端序列(19#引物扩增区)均存在不同程度的甲基化现象。其中,COX3基因的起始区序列仅在受精卵孵化期和幼体期存在甲基化现象,COX3基因3′端序列在除受精卵外的其他3个时期均存在甲基化现象,且以上甲基化率随个体发育阶段的提高呈下降趋势;ND5基因5′端序列在4个不同发育期均存在甲基化现象,但甲基化率无显著差异。甲基化主要发生在COX3基因和ND5基因位点上,推测脊尾白虾在生长发育过程中通过调节线粒体基因组甲基化来调节机体能量代谢过程。本研究首次从整个发育周期角度探讨了线粒体基因组甲基化特征,以期为进一步揭示甲壳类生长发育相关遗传机制提供帮助。     

凡纳滨对虾核自身抗原精子蛋白基因克隆及其表达

利用cDNA末端快速扩增技术克隆出凡纳滨对虾(Litopenaeus vannamei)核自身抗原精子蛋白(NASP)基因,NASP基因c DNA全长2258 bp,其中包括92 bp 5′端非编码区,147 bp 3′端非编码区及2019 bp开放阅读区,编码673个氨基酸,预测分子量为74.18 k D。该序列已提交至Gen Bank,序列号为KT274811。在NCBI上进行序列比对后发现,其与斑节对虾(Penaeus monodon)NASP序列相似性最高,为90%。采用荧光定量PCR方法测定了不同发育时期卵巢与肝胰腺中NASP m RNA相对表达水平,结果表明,NASP在卵巢中的表达量高于肝胰腺中的表达量,且在Ⅱ期表达量最高,Ⅲ期表达量最低。此外,通过构建系统进化树比较了凡纳滨对虾NASP与其他物种间的遗传距离。实验结果为进一步研究该基因在凡纳滨对虾卵巢发育中的作用提供了依据。     

中华绒螯蟹RXR基因全长cDNA克隆及表达分析

维甲类X受体(retinoid X receptor,RXR),属于核受体超家族(nuclear receptor super family)成员,是一种重要的调控因子,对甲壳动物生长发育和繁殖具有十分重要的作用。本研究根据陆地蟹 RXR基因保守区序列设计上下游引物,采用逆转录聚合酶链反应(RT-PCR)以及cDNA末端快速扩增(RACE)技术克隆中华绒螯蟹(Eriocheir sinensis)RXR基因并进行各组织间的表达分析。序列分析表明中华绒螯蟹RXR cDNA全长1517bp,编码433个氨基酸。经BLASTn和BLASTx分析表明,中华绒螯蟹RXR氨基酸序列与陆地蟹RXR基因的氨基酸序列相似性最高,为96%。系统进化树分析显示,中华绒螯蟹RXR的氨基酸序列与陆地蟹RXR聚为一支。荧光定量PCR结果显示,RXR在所有检测的组织中均有表达,且在中华绒螯蟹Y器官中表达量最高,肝胰腺、鳃和肌肉中有少量表达,心脏、胃、肠、胸神经节和脑神经节中微量表达。在中华绒螯蟹不同蜕皮时期中,Y器官中RXR表达量在D期最高,与AB期、C期呈显著性差异（P＜0.05）;肌肉中RXR在AB期表达量最高,与其他蜕皮时期呈显著性差异（P＜0.05）;肝胰腺和鳃中RXR在AB期表达量最高,E期表达量最低,但各蜕皮时期表达量之间差异不显著。     

水体pH对中华绒螯蟹幼蟹蜕壳生长及其相关基因表达的影响

为了解养殖水体p H值对中华绒螯蟹(Eriocheir sinensis)蜕壳生长及其相关基因表达的影响,在试验室水槽条件下,设定p H=7、8、9、10(均无水草)为实验组,以种植适量水草(p H值为8.2~9.1)为对照组,研究中华绒螯蟹幼蟹(均重0.43 g±0.09 g)在不同p H值水体中饲养一个蜕壳周期(37 d)的蜕壳生长及其相关基因(蜕皮抑制激素MIH,蜕皮激素受体EcR,维甲类X受体RXR,胰岛素样生长因子IGF2)表达情况。结果显示,对照组河蟹的成活率和增重率均显著高于实验组,其中,p H10的河蟹成活率和增重率最低。荧光定量PCR检测显示,MIH、EcR和RXR基因在不同p H水体的表达存在显著差异,IGF2基因在不同p H水体中的表达无显著差异;不同p H水体幼蟹的增重率与成活率之间存在显著正相关,EcR、RXR与IGF2基因相互间的表达量存在显著正相关,表明这三个基因的表达具有协同作用。通过对成活率、增重率和基因表达的综合分析认为幼蟹生长的最适p H为8~9。     

Intraspecific diversity of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Harvey) Suringar (Laminariales,Phaeophyta) from Japan,China and Korea,based on the <Emphasis Type="Italic">cox1</Emphasis> gene and ITS2 sequences

As a trial to develop a method of authenticating the place of origin of circulated Undaria pinnatifida products, we investigated their intraspecific genetic diversity using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) and the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA (rDNA) sequence. Four dried U. pinnatifida products labeled with their origins (one from Japan, one from China and two from Korea), natural plants collected from three locations (two from Japan and one from China), and cultivated plants collected from two locations (one from Japan and one from China) were used in the present study. The amplified fragments of cox1 were 664 bp in length, and the aligned sequences were highly homologous. Among the nine sequences, no insertions or deletions were found and six substitution positions were detected, and they were classified into five haplotypes. In contrast, multiple highly variable regions were found in ITS2, and some of them carried a restriction site for Mbo II. Polymerase chain reaction-restriction fragment length polymorphism analysis showed different restricted profiles among the tested samples. The availability of molecular markers for authenticating food products of U. pinnatifida is discussed.     

浅色黄姑鱼线粒体16S rRNA基因片段序列特征分析

PCR扩增100个浅色黄姑鱼个体的线粒体16SrRNA基因片段序列,得到大约620bp的扩增产物。将其中5个个体的扩增产物进行测序和同源比对,得到468bp可供比对分析的片段。比对结果表明,5条序列包括两个单倍型,两个单倍型之间有1个碱基突变。PCR-RFLP分析结果显示,两个种群的100个样品中98%的个体为其中一种单倍型,只有2%的个体呈另一种单倍型,表明这两个种群的遗传多样性较低。两个单倍型平均碱基组成为:T22.0%,C26.3%,A29.8%,G21.9%,GC含量平均为48.2%。与GenBank中石首鱼科7属9种的11条同源序列比对,得到429个比对位点,其中包括69个简约信息位点、55个单突变子和16个插入/缺失位点。聚类分析显示,浅色黄姑鱼与黄姑鱼亲缘关系较近,与形态分类相符。     

Intestinal microbiota of mangrove red snapper (Lutjanus argentimaculatus Forsskål, 1775) reared in sea cages

Using polymerase chain reaction amplification of 16S rDNA coupled to denaturing gradient gel electrophoresis (DGGE) and sequencing of isolated amplicons, we investigated the microbiota of the intestinal digesta and mucosal surface in mangrove red snapper cultured in a cage aquaculture area in Daya Bay. A total of 14 sequences were characterized by phylogenetic analysis. Among the bacterial species determinated from sequences, the γ‐Proteobacteria group (64.25%, nine species) dominated absolutely in fish intestines. Others belonged to Spirochaetes (14.3%, two species), Cyanobacteria (14.3%, two species) and Firmicutes (7.15%, one species). However, the bacteria were identified as uncultured accounting for 28.6% (four species). The apparent bacterial richness (calculated as the numbers of DGGE bands) was significantly higher in digesta than that in mucosal tissue samples (P<0.05). There existed five dominant individual populations including one unknown species of Firmicutes, Arthrospira sp., Vibrio metschnikovii, Vibrio harveyi and Vibrio sp. in intestinal digesta, and in contrast, only three dominant individual populations including Vibrio natriegens, V. harveyi and Vibrio sp. in intestinal mucosal surface. The results indicated that the microbiota in intestinal digesta was significantly different from that in mucosal surface.     

我国不同地理群体栉江珧遗传多样性及系统发生分析

利用通用引物对5个栉江珧野生群体(山东长岛、山东日照、山东文登、广东湛江和海南海口) 28S rRNA 和COI 基因片段进行扩增测序,分别得到983bp和623bp的片段,基于28S rRNA序列分析系统发生的结果表明,栉江珧与Atrina vexillumju具有较近的遗传关系。基于COI基因序列的遗传多样性分析结果显示,山东文登群体具有最高的遗传多样性水平。AMOVA分析显示,群体遗传分化系数为0.132 3(P<0.001),说明栉江珧遗传变异主要来源于群体内的变异。由28S rRNA和COI基因聚类分析结果推测,由于地理隔离,我国栉江珧南北方群体可能早已分化为不同亚种。这些数据为我国栉江珧种质资源保护和利用补充了分子生物学资料。     

Detection of infectious hemorrhagic disease of domestic rabbits (rabbit viral hemorrhagic disease) in wild rabbits (Oryctolagus cuniculus)

The occurrence of Rabbit Viral Hemorrhagic Disease of domestic rabbits in the population of wild rabbits in Germany was proven through detection of antigen (hemagglutination) in spleen and liver in one animal and through detection of specific antibody in another animal. There was no epizootic connection between the two animals (different locations). The necropsy and histopathologic findings are presented from five wild rabbits which died from RVHD.     

中国近海与日本近海白姑鱼线粒体细胞色素b基因全序列比较分析

对中国近海和日本近海共19尾白姑鱼(Pennahia argentata)线粒体细胞色素b(Cytochrome b,Cytb)基因全序列进行扩增与测定,白姑鱼个体的Cytb基因总长度均为1141bp。序列分析结果显示:Cytb基因全序列中共检测到40处核苷酸替代,全部为同义替换,且主要来自密码子第三位点。19尾白姑鱼个体共定义了14种单倍型。核苷酸组成分析表明:白姑鱼Cytb全序列的鸟嘌呤(G)含量较低,在第三密码子位点上表现得尤为明显。基于邻接法、最大简约法、最大似然法和贝叶斯法构建的系统树结果基本一致,中国近海和日本近海的白姑鱼明显分为2个单系群,两组群间的净遗传距离为0.019,基于Cytb2%/百万年分子钟计算,其分化时间约为95万年,中、日近海白姑鱼分化事件发生于晚更新世(Late Pleistocene)。本研究旨在探讨中、日近海白姑鱼组群的分类地位的遗传学依据,为白姑鱼渔业资源的管理与保护提供基础参考。     

Relationships between intracellular bacteria and the bivalve killer dinoflagellate Heterocapsa circularisquama (Dinophyceae)

ABSTRACT: Relationships between the growths of five different clonal strains of Heterocapsa circularisquama and intracellular bacteria were investigated using culture experiments. Although each H. circularisquama strain culture was established from one cell by repeated and careful washings with micropipettes, intracellular and extracellular bacteria in each strain culture were still observed under a epifluorescence microscope using the DAPI (4',6-diamidino-2-phenylindole) staining technique. The extracellular bacteria are derived, presumably, from the inside of algal cells after the death and collapse of algal cells in culture. Using an electron microscope, bacteria were constantly observed in the cytoplasm and food vacuoles of H. circularisquama cells. The growth of five algal strains containing bacteria was compared with that of a bacteria-free strain using culture experiments under combined conditions of five different light intensities and five different strengths of culture medium. Bacteria showed no significant effect on the growth or survival of the algal cells. During the algal exponential growth phase, the intracellular bacterial cell numbers per algal cell decreased, whereas the total bacterial cell density in each algal culture increased. Final cell yield (total number) of the intracellular and extracellular bacteria varied considerably according to the algal strains. These results suggest that the intracellular bacteria of H. circularisquama grow or survive depending on the host alga, and that the alga can grow independently.     

Presence of viruses in wild eels Anguilla anguilla L,from the Albufera Lake (Spain)

A virological analysis was conducted on wild eels from the Albufera Lake (Spain). A total of 179 individuals at different growth stages were collected in two different surveys (2004 and 2008). Presence of anguillid herpesvirus (AngHV‐1), aquabirnavirus and betanodavirus was confirmed by PCR procedures in both surveys, although the number of detections was clearly higher in 2008 (83% of the eels analysed resulted positive for virus presence). AngHV‐1 was the viral agent most frequently detected, followed by aquabirnaviruses. Betanodaviruses were detected by the first time in wild eels, and although the detections were only made by nested PCR, high percentage of positives were achieved. In addition, in 2008, seven aquabirnaviruses were isolated. Phylogenetic analysis performed using partial sequences of both genomic segments of aquabirnaviruses indicated that the seven isolates could be typed as WB (genogroup I) on the basis of segment A sequences, but when segment B was used six of them clustered with C1 strain (genogroup V) and one was typed as Ab (genogroup II). These results indicate natural reassortment between different strains of aquabirnaviruses in the eels. Although betanodaviruses were not isolated in cell culture, the analysis of the sequence of the nested PCR product indicated that they clustered with SJNNV genotype. The diversity of viral agents and the high level of viral detections suggest that viral infections may play a more prominent role in the decline of the European eel than initially thought.