翘嘴鳜miR-222的表达特征（英文）

为了解翘嘴鳜mi R-222的时空表达规律,研究利用实时荧光定量PCR的方法检测mi R-222在翘嘴鳜不同组织、胚胎发育及胚后发育中的相对表达丰度。研究结果显示,mi R-222在肌肉相关的组织中表达较高,特别是在成年翘嘴鳜的白肌中表达最高;胚胎发育阶段结果显示,mi R-222在胚胎发育的2细胞期就有表达,而表达量在心动期达到最高。不同组织及不同发育阶段的差异性表达结果表明,mi R-222很可能参与调控鳜鱼肌肉的生长发育。为研究合成代谢过程中mi R-222在肌肉生长调控中的表达规律,通过对翘嘴鳜幼鱼在饥饿一周后饱食一餐的实验处理下,利用实时荧光定量的方法测定mi R-222在骨骼肌中的相对表达变化。结果显示,mi R-222的表达量在恢复喂食后的1h显著上升(P0.05),表明mi R-222很可能是调节鱼类骨骼肌生长过程中,参与快速应答信号系统的一类mi RNA。研究为mi R-222在鱼类发育中的调控作用提供一些理论依据。     

MRFs家族基因在翘嘴鳜成体不同组织及器官中的表达特征

生肌调节因子(MRFs)家族成员包括MRF4、Myf5、Myogenin和MyoD,是肌肉形成的关键控制因素,其作为一种转录因子在肌肉的发育分化过程中发挥重要作用。本研究通过RT-qPCR方法分析MRFs家族基因在翘嘴鳜成体中不同组织及器官的表达情况,阐明其在肌肉组织中的特异性表达。结果显示:MRFs家族基因在成体翘嘴鳜肌肉、心脏、肝脏、脾脏、肾脏、肠道和脑组织及器官中均检测到表达,且在肌肉中的表达量显著高于其他组织及器官中的表达量(p<0.05)。为研究生肌调节因子在翘嘴鳜肌肉发育过程中的作用提供了基础资料。     

翘嘴鳜性腺型芳香化酶基因CYP19a的克隆及表达研究

采用同源克隆与SMART RACE技术首次分离和克隆了翘嘴鳜性腺型芳香化酶基因CYP19a的c DNA全长,并通过实时荧光定量PCR(real-time quantitative PCR,q PCR)技术分析了其在翘嘴鳜成体不同组织及性腺不同发育时期的表达特点。序列分析结果显示,CYP19a基因全长1 873 bp(Gen Bank登录号为KX911988),其中开放阅读框长1 581 bp,编码526个氨基酸。通过多序列比对,发现该基因编码的蛋白质具有P450arom A特定的功能保守序列,包括I-螺旋区、Ozol肽区、亚铁血红素结合区域以及芳香化酶特异性结合区域;同时,CYP19a基因序列的同源性分析结果显示,其在脊椎动物中较为保守。此外,q PCR检测结果显示,CYP19a基因在翘嘴鳜成体组织中的表达存在显著差异(P0.05),主要在性腺中表达,其次在脑和肝脏有少量表达;而且CYP19a基因在繁殖期性腺中的表达量显著低于非繁殖期性腺中的表达量(P0.05)。以上研究表明,CYP19a基因在鱼类性腺形成及发育过程中具有重要作用。     

翘嘴鳜microRNA转录组分析及生长相关miRNA鉴定

MicroRNAs(mi RNAs)是一类内源性、长度约22 nt的非编码小RNA,通过转录后调控基因的表达水平、参与生物体的生长与发育等多种生理过程。翘嘴鳜是我国淡水经济鱼类,由于长期人工繁殖忽视亲本选育,导致鳜种质退化,出现生长速度及抗病力下降等问题。本研究中,我们以生长存在显著差异的慢长组和快长组翘嘴鳜肌肉为材料,通过HiSeq 2000深度测序技术和生物信息学分析miRNA转录组,鉴定出433个已知miRNAs并分析miRNAs种子编辑情况。Expdiff方法结合qPCR验证试验,证实了4个表达显著差异的mi RNAs:miR-122、miR-192、miR-451、let-7j-5p。对差异miRNAs进行靶基因预测并通过KEGG Pathway显著性富集分析,发现差异miRNA参与生长、发育、代谢、信号转导等途径,其中Wnt信号通路等在肌肉的生长发育过程起重要作用。本研究为进一步开展miRNA-靶基因的交互作用、了解其对鳜鱼生长的调控机制奠定了基础,并可为鳜鱼分子选育种提供候选基因。     

翘嘴鳜per1 mRNA表达量分析中采用的内参基因稳定性比较

为筛选生物钟核心基因per1表达定量中的相对稳定性最好的内参基因,本研究取翘嘴鳜成鱼心脏、肝脏、肾脏、脑、红肌、白肌、肠、眼和脾等九个组织为研究对象,选取GAPDH、18S rRNA、β-actin、rps29、RPL13a、B2M和EF1a为内参基因,采用实时荧光定量PCR(qRT-PCR)对per1基因mRNA表达水平进行检测分析。研究结果表明18S rRNA和GAPDH的平均稳定值M最低,相对表达量最稳定。以18S rRNA和GAPDH为内参基因时分析发现per1基因表达量在肝脏中最高。本研究为在鱼类per1 mRNA表达检测过程中选用稳定的内参基因提供了实验和理论参考。     

翘嘴鲌Sox9基因的克隆及CpG岛甲基化与基因表达的关系

为了揭示翘嘴鲌(Culter alburnus)性别决定与分化的作用机制, 进而更好地发展性别控制育种技术, 研究重点分析了Sox9基因在翘嘴鲌性腺分化过程中的作用。通过RT-PCR和RACE方法获得了翘嘴鲌2个旁系同源基因Sox9a和Sox9b的cDNA序列: Sox9a全长1642 bp, 编码458个氨基酸; Sox9b全长1673 bp, 编码456个氨基酸。序列分析表明两者相似度达到73.95%, 编码HMG盒区域极其保守。蛋白质次级结构预测显示Sox9a和Sox9b除了保守的HMG盒结构域外, 还存在2个核定位信号; 两者的三维结构都存在多个螺旋结构。系统进化树分析发现翘嘴鲌Sox9a与罗非鱼关系最近, 但Sox9b形成单独的一支。利用实时荧光定量PCR技术分析了翘嘴鲌Sox9a和Sox9b基因在各成体组织中的表达水平, 结果显示Sox9a在脑和精巢中表达量最高, 其次是肌肉、鳍条、眼睛和卵巢, 在肾脏、脾脏、肝脏中相对较低; Sox9b只在脑、鳍条、眼睛和精巢中检测到一定水平的表达。通过重亚硫酸氢盐DNA测序方法分析了翘嘴鲌性腺组织Sox9a启动子CpG岛甲基化修饰模式, 结果显示在精巢中CG位点几乎不发生甲基化, 然而卵巢中的甲基化程度非常高。这些结果表明启动子CpG甲基化可以调控Sox9a的性别异形表达, 表观遗传修饰在翘嘴鲌性腺发育过程中可能具有重要的生物学功能。     

翘嘴鳜NADPH氧化酶2个催化亚基cDNA的克隆和表达特征

吞噬细胞NADPH氧化酶能生成用于清除病原微生物的活性氧(reactive oxygen species, ROS),在机体的防御体系中起着非常重要的作用.本文利用RT-PCR结合RACE-PCR的方法,克隆到翘嘴鳜NADPH氧化酶的催化亚基gp91phox和p22phox的cDNA全长.并研究两者在正常的翘嘴鳜和注射了柱状黄杆菌灭活菌苗（FKG4）的翘嘴鳜组织中的表达模式.结果表明,gp91phox基因cDNA序列全长2 037 nt,开放阅读框长度为1 698 nt,翻译成565个氨基酸;p22phox 基因cDNA序列全长1 296 nt,开放阅读框561 nt,翻译成186个氨基酸.将这2个亚基推导的氨基酸序列与人的对应亚基相比,相似性分别为68.7%和60.8%,且具有相似的结构域和功能域,说明翘嘴鳜与人的NADPH氧化酶具有相似的功能活性.半定量PCR分析显示,在翘嘴鳜血液、脑、心脏、肾、肝、脾、胸腺等11种组织中均能检测到gp91phox和p22phox的基因表达.经FKG4免疫后,gp91phox在翘嘴鳜血液、头肾和脾3种组织中的表达量显著上升,p22phox在头肾和脾2种组织中的表达量显著上升.由此推断,NADPH氧化酶可能参与了机体的抗菌免疫应答.     

miR-196b-5p促进成肌细胞增殖分化

MicroRNA (miRNA)是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子，在动植物中参与转录后基因表达调控。大量研究表明，miRNA调控骨骼肌的发育，主要表现在肌卫星细胞的激活及增殖、分化、肌管的形成等生物学过程。本实验室前期对大白猪背最长肌(longissimus dorsi,LD)和比目鱼肌(soleus muscle,Sol)进行miRNA测序，筛选鉴定到一个在不同骨骼肌中差异表达并且序列高度保守的miR-196b-5p，目前miR-196b-5p在骨骼肌方面的研究尚未见报道。本研究进一步设计合成miR-196b-5p mimics和inhibitor对C2C12细胞进行miR-196b-5p过表达及干扰表达，利用蛋白免疫印迹、实时荧光定量PCR检测、流式细胞术、免疫荧光染色等方法探究miR-196b-5p对成肌细胞增殖分化的影响，并利用生物信息学预测和双荧光素酶报告系统鉴定了miR-196b-5p的靶基因。结果显示，过表达miR-196b-5p显著增加细胞周期基因Cyclin B、Cyclin D和Cyclin E的mRNA和蛋白表达水平(P<0....     

在不同养殖背景下鳜mc1r组织表达差异和启动子转录调控分析

为探究鳜(Siniperca chuatsi)黑素皮质素1受体基因mc1r在不同养殖背景下的组织表达差异及转录调控机制,研究以在黑白养殖背景下产生的深、浅色体表鳜为研究对象,通过实时荧光定量PCR测定mc1r基因的组织表达差异;其次以鳜基因组DNA为模板,通过PCR扩增mc1r基因5′侧翼区不同长度的缺失片段并克隆至pGL3-Basic载体,之后将重组质粒转染到HEK 293T细胞,检测双荧光素酶的相对活性并预测其存在潜在的转录因子。结果显示,黑色背景养殖所产生的深色体表鳜与野生型更为相似; RT-qPCR检测显示深色体表鳜mc1r在背部皮肤和腹部皮肤表达量最高,而浅色体表鳜脑中表达量最高;双荧光素酶活性检测发现,构建的5个缺失片段启动子活性之间存在显著差异,且-159∽-+292启动子活性最高,推测其为mc1r核心启动子区域,并且通过在线软件预测出存在MITF、SP1等转录因子。研究克隆了鳜mc1r基因启动子区域并进行了转录活性分析,并且对潜在的转录因子进行了预测,为鱼类mc1r基因分子机制表达调控及表皮颜色可控性研究提供了理论依据。     

Characterization and ontogenetic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi

Three full-length complementary DNA (cDNA) clones were isolated encoding the skeletal myosin light chain 1 (MLC1; 1237 bp), myosin light chain 2 (MLC2; 1206 bp) and myosin light chain 3 (MLC3; 1079 bp) from the fast white muscle cDNA library of mandarin fish Siniperca chuatsi. The sequence analysis indicated that MLC1 and MLC3 were not produced from differentially spliced messenger RNAs (mRNA) as reported in birds and rodents but were encoded by different genes. The MLC2 encodes 170 amino acids, which include four EF-hand (helix-loop-helix) structures. The primary structures of the Ca(2+)-binding domain were well conserved among the MLC2s of seven other fish species. The ontogenetic expression analysis by real-time PCR showed that the three light-chain mRNAs were first detected in the gastrula stage, and their expression increased from the tail bud stage to the larval stage. All three MLC mRNAs showed longitudinal expression variation in the fast white muscle of S. chuatsi, especially MLC1 which was highly expressed at the posterior area. Taken together, the study provides a better understanding about the MLC gene structure and their expression pattern in muscle development of S. chuatsi.     

鳜早期发育阶段骨骼肌纤维的增生与肥大生长

骨骼肌是鱼体的主要组成部分,也是衡量鱼体生长发育的重要指标.为了解鳜(Siniperca chuatsi)早期发育阶段骨骼肌纤维的生长发育特征,通过制作孵化后1 ～41日龄个体骨骼肌背右侧第一肌节石蜡切片,利用图像分析软件统计该肌节中肌纤维的数目和面积,分析了鳜骨骼肌纤维的增生和肥大生长特征.结果表明,鳜早期发育阶段骨...     

两种鳜病毒的共感染现象及引起感染细胞的超微变化

借助细胞培养和电镜技术,揭示了鳜球形病毒(Siniperca chuatsi spherical virus,SCSV)与鳜弹状病毒(Siniperca chuatsi rhabdovirus,SCRV)在草鱼鳍细胞(Grass carp fins,GCF)中共感染的现象。在筛选到敏感鱼类细胞系和建立了 鳜病毒体外增殖系统的基础上,取息典型病毒感染出血症的鳜组织,制备组织悬液,接种到GCF细胞中传代培养, 在攻毒后间隔不同时间收集细胞,对攻毒细胞的超薄切片进行电镜观察。揭示两种形态的鳜病毒可在同一个GCF 细胞中增殖,并描述和分析了病毒复制引起感染细胞的超微病变。本研究结果有助于阐明鱼类重要病毒病害的发 生过程及致病机理。     

MicroRNA-499 Expression Distinctively Correlates to Target Genes sox6 and rod1 Profiles to Resolve the Skeletal Muscle Phenotype in Nile Tilapia

A class of small non-coding RNAs, the microRNAs (miRNAs), has been shown to be essential for the regulation of specific cell pathways, including skeletal muscle development, maintenance and homeostasis in vertebrates. However, the relative contribution of miRNAs for determining the red and white muscle cell phenotypes is far from being fully comprehended. To better characterize the role of miRNA in skeletal muscle cell biology, we investigated muscle-specific miRNA (myomiR) signatures in Nile tilapia fish. Quantitative (RT-qPCR) and spatial (FISH) expression analyses revealed a highly differential expression (forty-four-fold) of miR-499 in red skeletal muscle compared to white skeletal muscle, whereas the remaining known myomiRs were equally expressed in both muscle cell types. Detailed examination of the miR-499 targets through bioinformatics led us to the sox6 and rod1 genes, which had low expression in red muscle cells according to RT-qPCR, FISH, and protein immunofluorescence profiling experiments. Interestingly, we verified that the high expression of miR-499 perfectly correlates with a low expression of sox6 and rod1 target genes, as verified by a distinctive predominance of mRNA destabilization and protein translational decay to these genes, respectively. Through a genome-wide comparative analysis of SOX6 and ROD1 protein domains and through an in silico gene regulatory network, we also demonstrate that both proteins are essentially similar in vertebrate genomes, suggesting their gene regulatory network may also be widely conserved. Overall, our data shed light on the potential regulation of targets by miR-499 associated with the slow-twitch muscle fiber type phenotype. Additionally the results provide novel insights into the evolutionary dynamics of miRNA and target genes enrolled in a putative constrained molecular pathway in the skeletal muscle cells of vertebrates.     

Differential microRNA Expression in Fast- and Slow-Twitch Skeletal Muscle of Piaractus mesopotamicus during Growth

Pacu (Piaractus mesopotamicus) is a Brazilian fish with a high economic value in pisciculture due to its rusticity and fast growth. Postnatal growth of skeletal muscle in fish occurs by hyperplasia and/or hypertrophy, processes that are dependent on the proliferation and differentiation of myoblasts. A class of small noncoding RNAs, known as microRNAs (miRNAs), represses the expression of target mRNAs, and many studies have demonstrated that miR-1, miR-133, miR-206 and miR-499 regulate different processes in skeletal muscle through the mRNA silencing of hdac4 (histone deacetylase 4), srf (serum response factor), pax7 (paired box 7) and sox6 ((sex determining region Y)-box 6), respectively. The aim of our work was to evaluate the expression of these miRNAs and their putative target mRNAs in fast- and slow-twitch skeletal muscle of pacu during growth. We used pacus in three different development stages: larval (aged 30 days), juvenile (aged 90 days and 150 days) and adult (aged 2 years). To complement our study, we also performed a pacu myoblast cell culture, which allowed us to investigate miRNA expression in the progression from myoblast proliferation to differentiation. Our results revealed an inverse correlation between the expression of the miRNAs and their target mRNAs, and there was evidence that miR-1 and miR-206 may regulate the differentiation of myoblasts, whereas miR-133 may regulate the proliferation of these cells. miR-499 was highly expressed in slow-twitch muscle, which suggests its involvement in the specification of the slow phenotype in muscle fibers. The expression of these miRNAs exhibited variations between different development stages and between distinct muscle twitch phenotypes. This work provides the first identification of miRNA expression profiles in pacu skeletal muscle and suggests an important role of these molecules in muscle growth and in the maintenance of the muscle phenotype.     

鳜鱼病毒病原的检出及组织病理分析

经对患暴发性传染病的鳜组织及病变组织超微切片的电镜观察,在细胞质,核及间隙中都有病毒颗粒,对该病毒进行了分离提纯,这是一种直径约为２８０ｎｍ球形病毒,将粗提病毒液接种到培养细胞中,会引起细胞病变;病毒通过人工感染健康鳜,会引起鳜发病和死亡,对患病獗进行解剖和组织病理观察,表明病鳜的５种器官都发生了病变,研究结果表明分离的鳜病毒（ＳｉｎｉｐｅｒｃａｃｈｕａｔｓｉＶｉｒｕｓ,ＳＣＶ）是引起鳜流４行的病     

γ-氨基丁酸对鳜摄食和食欲的影响

为阐明γ-氨基丁酸(GABA)对鳜(Siniperca chuatsi)摄食和食欲的影响, 对鳜脑室注射生理盐水和不同剂量的GABA(50、125、500和2000 μg)。结果显示, 注射125 μg GABA组的鳜在2h内摄食量显著升高。实时荧光定量PCR (RT-qPCR)结果显示, 注射125 μg GABA 0.5h后, 鳜鱼脑中AgRP和NPY mRNA表达量上调, CART和POMC mRNA表达量下调, 都和鳜摄食量增加相一致。相比于对照组, 注射GABA后Leptin-R的mRNA表达量在0.5h和2h都有显著下降。这些结果表明GABA可能通过leptin的信号通路来影响食欲, 进而影响摄食量。研究结果可以为GABA在水产饲料中的应用提供理论依据。