Use of suppressive subtractive hybridization and cDNA arrays to discover patterns of altered gene expression in the liver of dihydrotestosterone and 11-ketotestosterone exposed adult male largemouth bass (Micropterus salmoides)

In this study male largemouth bass (LMB) were exposed to the naturally occurring androgens, dihydrotestosterone (DHT) or 11-ketotestosterone (11-KT) in order to identify genes that are differentially regulated by these steroid hormones. Using subtractive hybridization on livers of fish treated with DHT against vehicle control, many novel LMB genes were cloned. These genes were added to our gene library and arrayed. Six genes were up-regulated and five were down-regulated by both androgens. But, each androgen also regulated specific genes. One gene that was identified as a potential androgen marker was spermidine-spermine-N¹-acetyltransferase that was up-regulated by both androgens. Determining which genes are responsive to natural androgens will help to identify biochemical pathways that are impacted.     

Perturbation of gene expression and steroidogenesis with in vitro exposure of fathead minnow ovaries to ketoconazole

Ketoconazole is a fungicidal drug that inhibits function of cytochrome P450s in the synthesis of steroids. To examine if inhibition of P450 function affects gene expression in a dynamic manner, we conducted in vitro exposures of ovary tissue from fathead minnows (Pimephales promelas) to 0.5 microM ketoconazole to investigate effects on steroid production and gene expression over time. Expression of four key steroidogenesis genes was examined at 1, 6, and 12h of exposure. 11 beta- and 20 beta-hydroxysteroid dehydrogenases were down regulated at 1h and Cytochrome P450 17 was down-regulated at 12h, consistent with the absence of steroid production. In contrast, cytochrome P450 19A was up-regulated at 6h, indicating feedback regulation. Microarray analysis of 12h exposures indicated enrichment of biological processes involved in neurotransmitter secretion, lymphocyte cell activation, sodium ion transport, and embryonic development. These data suggest that, with the exception of cytochrome P450 19A, these steroid metabolic genes are regulated in a feed forward manner and that the effects of ketoconazole may be broader than anticipated based on the mechanism of action alone.     

北极细菌Pseudoalteromonas sp.A2对H2O2胁迫的转录组分析

海水中的高溶解氧浓度、低温和UV辐射导致了氧胁迫是北极海洋细菌面临的主要胁迫因素之一。本文对北极细菌Pseudoalteromonas sp.A2对H₂O₂导致的氧胁迫的应答特征进行了转录组测序和基因差异表达的比较分析,以期发现与氧胁迫相关的关键功能基因。研究表明,与对照组相比,1 mmol/L的H₂O₂可导致菌株A2转录组的很大变化,包括109个基因的显著上调与174个基因的显著下调。COG分析表明,在功能已知的基因中,与转录后修饰、蛋白质转换和分子伴侣相关的基因大部分显著上调,而与氨基酸运输和代谢等相关基因则大部分显著下调。值得指出的是,有18个与鞭毛相关的基因和4个与热激蛋白相关的基因显著上调;同时,有9个与细胞色素和细胞色素氧化酶相关的基因和5个与TonB依赖受体相关的基因显著下调。在GO分析表明具有抗氧化活性的18个基因中,只有1个基因的表达显著上调,而有2个显著下调。简言之,RNA-Seq表明,除了传统的抗氧化基因和应激蛋白外,鞭毛相关基因和功能未知基因在菌株Pseudoalteromonas sp.A2的氧胁迫适应性中起着重要作用。该转录组分析和氧胁迫相关基因的发现有助于了解北极细菌的氧胁迫适应机制。     

Differential gene expression in Poecilia vivipara exposed to diesel oil water accommodated fraction

Diesel fuel is a potential contaminant of estuarine and mangrove areas, particularly because it is the main fuel used in small boats and larger vessels. The aim of this work was to identify genes differentially expressed in the liver of Poecilia vivipara (Guppy) exposed to 10% diesel fuel water accommodated fraction (WAF), employing the subtractive suppressive hybridization (SSH) method. The results showed 27 differentially expressed gene fragments, 12 up-regulated and 15 down-regulated. Among the up-regulated genes were CYP1A, UDPGT1a, ABCC4, Methyltransferase and Apolipoprotein A1. Down-regulated genes included Vitellogenins, C1 Inhibitor and Complement Component 3c. The identified genes are associated with different metabolic functions like biotransformation, membrane transport and immune system, indicating the susceptibility and/or molecular responses of this organism to the toxic effects elicited by diesel fuel WSF.     

Primary molecular basis of androgenic gland endocrine sex regulation revealed by transcriptome analysis in Eriocheir sinensis

In crustaceans, the male sexual dif ferentiation and maintenance are specially regulated by androgenic gland(AG). However, little is known about the genes involved in the regulation process.RNA-Seq was performed on AG with ejaculatory duct(AG_ED) and ejaculatory duct(ED) as control in Eriocheir sinensis, one of the most important economic and ?shery crabs with typically sex dimorphism. A total of 925 unigenes were identi?ed as dif ferentially expressed genes(DEGs) and the expression of nine genes randomly selected was con?rmed by qRT-PCR. 667 unigenes were up-regulated in AG_ED, being supposed to be AG preferential genes. Among them, the full length of i nsulin-like androgenic gland factor( IAG) cDNA named as Es-IAG was obtained as a logo gene of AG, which together with the genes i nsulin-like receptor( INR), and s ingle insulin binding domain protein( SIBD), might constitute the sex regulation pathway. Several sex related genes were identi?ed, and their function will have to be investigated. Also,the identi?cation of j uvenile hormone epoxide hydrolase 1( JHEH1), ecdysteroid 22-hydroxylase( DIB) and e cdysone receptor( ECR) preliminarily clari?ed the molecular regulation mechanism of eyestalk-AG-testis axis, which plays important roles in molting and reproduction. The results will enhance our understanding for the molecular basis of the AG involved in male sex regulation in crabs.     

Identification of transcriptional effects of ethynyl oestradiol in male plaice (Pleuronectes platessa) by suppression subtractive hybridisation and a nylon macroarray

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.     

低温胁迫对军曹鱼幼鱼脂代谢相关基因表达的影响

将两组军曹鱼(Rachycentron canadum)幼鱼分别在常温(30.5±1.0)℃和低温(20.0±0.5)℃环境下饲养7 d,并于第1天、第4天、第7天3个时间点采集肝脏、肌肉和腹腔脂肪,利用实时荧光定量PCR(qRT-PCR)技术分析5个脂代谢相关基因的表达变化情况,以探究低温胁迫对军曹鱼脂质合成与分解代谢的影响。结果显示,第1天时,肝脏的肉碱脂酰基转移酶-1基因(cpt-1)、脂肪激素敏感脂肪酶基因(hsl)以及肌肉的cpt-1、hsl、单酰基甘油酯酶基因(mgl)等显著上调(p<0.05),肝脏、肌肉的乙酰辅酶A羧化酶基因(acc)和脂肪酸合成酶基因(fas)以及腹腔脂肪(IPF)5个脂代谢相关基因表达均显著下调(p<0.05);第4天时,肝脏的cpt-1、hsl、mgl和肌肉的hsl、mgl、acc、fas以及IPF的cpt-1、hsl、mgl、acc等表达上调(p<0.05),肝脏的acc、fas表达显著下调(p<0.05);第7天时,肝脏和IPF的cpt-1、hsl、mgl、acc和肌肉的hsl、mgl、acc等表达上调(p<0.05),肌肉cpt-1和肝脏fas表达显著下调(p<0.05)。结果表明,军曹鱼幼鱼在低温胁迫前期通过抑制脂合成代谢,促进肝脏和肌肉中的脂质水解,通过抑制腹腔脂肪的脂质分解来响应低温胁迫;在低温胁迫后期,军曹鱼幼鱼脂合成和分解代谢均显著提高,且利用脂肪酸提供能量的主要组织由前期的肝脏和肌肉转变为肝脏和腹腔脂肪。     

斑马鱼(Danio rerio)胚胎免疫反应: 补体基因在早期胚胎以及LPS 刺激胚胎中的表达

以模式生物斑马鱼(Danio rerio)为研究对象, 使用原位杂交及实时荧光定量PCR 方法, 对 斑马鱼补体基因(C3, C4, C9, Bf1, Bf2, Bf3)及其调节因子(RCA2.1, RCA2.2)在早期胚胎中的时空表达 模式及LPS 刺激后表达量的变化进行了研究。结果表明, 上述基因在胚胎早期(卵裂期至体节期)均 为泛表达, 从受精后24h 至幼鱼阶段特异性表达于肝脏及消化道等器官。对胚胎显微注射LPS 后, C3、C9、Bf1、Bf2、Bf3 基因在早期胚胎中表达上调, RCA2.1 基因表达下调, 这与斑马鱼成鱼受到 革兰氏阴性菌感染后补体分子(C3, C9, B 因子等)的反应类似, 表明其参与补体激活途径、溶膜途径 及补体调节, 提示斑马鱼可在胚胎发育早期构建补体系统, 参与急性期反应等免疫反应。     

高温胁迫下坛紫菜的数字基因表达谱研究

坛紫菜是潮间带重要的经济藻种,对高温、渗透压等逆境具有独特的调控机制。本文采用基于高通量测序的数字基因表达谱(DGE)技术研究了坛紫菜在高温胁迫下的基因表达差异,并分析其相应的响应方式;利用实时定量PCR技术对DGE部分数据进行验证;检测了其中较有代表性的应答基因hsp70的差异表达。结果显示,高温胁迫下坛紫菜中有256个unigene上调表达,以HSP、核糖体蛋白L12、延伸因子EF-Tu及部分光合作用相关基因为代表,3 820个unigene下调表达,主要为核酸、蛋白以及糖类等合成代谢相关基因。Gene Ontology分析表明,差异表达基因主要定位于质体等有膜细胞器,参与繁殖和发育过程,行使催化和连接酶活性的功能。Pathway分析显示,这些基因分布于107条pathway中。其中,下调表达基因最显著富集于mRNA监督和RNA转运途径,而上调表达基因部分富集于内质网的蛋白加工、RNA降解及光合作用途径。验证表明此次DGE结果具有较高准确性,hsp70基因对高温响应积极。综上所述,DGE结果反应出,在高温胁迫时,坛紫菜出现基础代谢减慢、合成速度下降、能量合成受阻、碳同化降低等现象,但光合作用前期未受影响,同时补救途径启动。     

Identification of transcriptional effects of ethynyl oestradiol in male plaice (Pleuronectes platessa) by suppression subtractive hybridisation and a nylon macroarray

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.     

溶藻弧菌体外刺激泥蚶(Tegillarca granosa)血细胞后早期转录组研究

泥蚶(Tegillarca granosa)是我国传统养殖贝类,细菌性疾病导致的死亡已成为制约泥蚶养殖业发展的重要因素,血细胞是泥蚶免疫防御的执行者,血细胞能直接吞噬异物,能通过包囊作用将异物包裹,血细胞还在炎症反应和伤口修复中发挥作用。采用RNA-seq测序技术研究了溶藻弧菌体外刺激后泥蚶血细胞3h和6h时转录组动态变化,与未受刺激血细胞相比,3h时1790个基因表达发生显著改变,包括1319个上调基因和471个下调基因;6h时3183个基因表达发生显著改变,包括2629个上调基因和554个下调基因。KEGG通路富集分析发现溶藻弧菌刺激后免疫相关多个信号通路或生物学过程发生显著改变,如吞噬体、细胞凋亡、蛋白酶体、内质网加工、局部黏附等。部分免疫相关基因表达丰度3h时变化不显著而6h时发生显著改变,部分免疫相关基因3h和6h时表达丰度均显著改变。研究结果为泥蚶血细胞早期免疫反应提供了新的见解,为泥蚶抗病机理研究提供了理论基础。     

凡纳滨对虾P23蛋白基因的筛选、表达和生物信息学分析

克隆凡纳滨对虾极端体重个体的组织差异表达基因P23基因并进行生物信息学分析, 为进一步研究该基因的功能以及凡纳滨对虾的分子选育等研究提供信息。以凡纳滨对虾雌虾极端体重个体腹部肌肉为实验材料, 利用抑制消减杂交(SSH)技术构建极大体重雌性个体和极小体重雌性个体腹部肌肉组织正反向消减cDNA文库:正库(以极大体重个体为试验组, 以极小体重个体为驱动组, L-S)和反库(以极小体重个体为试验组, 以极大体重个体为驱动组, S-L)消减cDNA文库, 并采用实时定量技术分析P23基因的组织表达规律, 利用生物信息学对其功能和结构进行预测。以β-actin为看家基因检测两个文库的消减效率分别为210和25, 实时定量技术分析结果表明P23基因在体重的调节中起上调作用。P23基因编码区序列全长495bp, 编码165个氨基酸, GenBank登录号:JF806619。生物信息学分析发现P23蛋白部分序列含有强疏水性, 无跨膜螺旋结构, 两种信号肽预测都显示P23含有信号肽。     

马氏珠母贝PfAQP4免疫功能初探

文章对马氏珠母贝水通道蛋白基因PfAQP4进行免疫功能分析。利用实时荧光定量分析免疫刺激和RNA干扰后PfAQP4及免疫相关基因的表达模式, 旨在为马氏珠母贝自然免疫研究提供新的资料。结果表明, 注射脂多糖LPS后, 在外套膜组织中PfAQP4 mRNA表达量在24h时显著上调(p<0.05), 在消化腺中PfAQP4 mRNA表达量先在12h时显著下调(p<0.05), 后在24h、36h和48h时显著上调(p<0.05)。注射聚肌胞poly(I:C)后, 在外套膜组织中PfAQP4 mRNA表达量在36h时和48h时显著上调(p<0.05), 在消化腺中PfAQP4 mRNA表达量在48h时显著上调(p<0.05)。RNA干扰PfAQP4表达后, 免疫相关基因CuZn-SOD在外套膜中的表达量显著下降(p<0.05), Pearson相关性分析显示PfAQP4与CuZn-SOD的表达存在显著正相关(r=0.818, p<0.001)。综上所述, PfAQP4参与马氏珠母贝的免疫应答。     

盐度对广盐型聚球藻K1生长及转录组的影响*

聚球藻是一类分布广泛、数量巨大的微微型浮游植物, 作为蓝藻的代表类群之一, 广泛分布于海洋以及河口区域, 并且具有丰富的色素多样性和遗传多样性。聚球藻根据盐度适应能力可划分为严格海洋型聚球藻和广盐型聚球藻。文章对分离自珠江口的聚球藻K1和南海寡营养海域的聚球藻YX02-1在不同盐度条件下的生长状况进行对比, 并根据ropC1标记基因分析K1和YX02-1的分类地位, 发现K1为广盐型聚球藻, 其在不同盐度下(10‰、13‰、18‰、25‰、33‰)均能生长; 而YX02-1为严格海洋型聚球藻, 在低于13‰的盐度下无法存活, 与其在河口的分布特征相一致。转录组分析显示, 低盐条件下广盐型聚球藻中合成渗透压调节分子的基因(ggpS、SPS、stpA)表达量明显下调, 而膜通道蛋白glzT基因的表达显著上调, 说明广盐型聚球藻耐低盐机制主要是通过减少细胞内渗透压相关小分子合成和增加膜通道蛋白, 提高小分子物质外排来达到细胞内外渗透压平衡。此外, 盐度也会影响广盐型聚球藻的光合作用和代谢水平, 其中参与光合作用的产能基因(ATPF0B、ATPF0A、ATPF1D、ATPF1A)和色素蛋白基因(cpcA、cpcB)表达量在低盐条件下均显著下降, 同时无机氮利用相关基因表达上调。低盐条件下, 渗透压相关小分子的需求减少, 可以使更多的碳源用于支持生长, 同时无机氮的吸收增强, 这两者可能是低盐条件下广盐型聚球藻具有较高生长的原因。     

Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis

A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus （ Litopenaeus ） setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.     

基于16S rDNA和看家基因序列分析技术的鲍鱼养殖水体弧菌种类的鉴定

采用TCBS选择性培养基从厦门某鲍鱼养殖场的养殖水体中分离到19株细菌.经16S rDNA序列分析,所有菌株均属于弧菌属（Vibrio）,且与最近似的弧菌模式种的同源性都在98.99%以上.由于弧菌种间16S rDNA序列相似度极高,无法仅靠16S rDNA序列比对来鉴定这些菌株到种的水平.16S rDNA序列的系统发育分析也显示,大多数菌株与弧菌模式种无法归为一簇,表明16S rDNA基因在弧菌种的分类鉴定上分辨率不高.进一步采用基于4种看家基因（rpo A、pyr H、gap A和top A）的多位点序列分析技术（MLSA）对分离到的弧菌菌株进行分类鉴定,结果显示19株海洋弧菌归于溶藻弧菌（Vibrio alginalyticus）与魔鬼弧菌（Vibrio diabolicus）2个种,表明这两种弧菌是该鲍鱼养殖场水体环境中的优势弧菌种,在鲍鱼养殖弧菌病害防治方面需要重点关注.此外,看家基因与16S rDNA基因的多态性分析表明,4种看家基因的多态位点比率均高于16S rDNA.4种看家基因串联后的多态位点比率高达41.1%,远高于16S r DNA基因的13.4%,表明看家基因相较于16S rDNA基因有着更高的分辨率,更适合于海洋弧菌的分类鉴定.     

褐斜鲽(Plagiopsetta glossa)线粒体基因组特征及重排机制研究

冠鲽科(Samaridae)隶属于鲽形目(Pleuronectiformes), 包含冠鲽属(Samaris)、沙鲽属(Samariscus)和斜鲽属(Plagiopsetta)。目前研究表明, 冠鲽(Samaris cristatus)和满月沙鲽(Samariscus latus)的线粒体基因组结构都有重排, 并且两者的基因数量也有差别。为检测斜鲽属鱼类中是否也有不同的特征结构, 我们选用褐斜鲽(Plagiopsetta glossa)作为代表种进行斜鲽属鱼类线粒体基因组特征分析, 同时与冠鲽属及沙鲽属鱼类的线粒体基因组特征进行对比。分析显示, 褐斜鲽的线粒体基因组全长为18723bp, 包括39个基因: 13个蛋白基因、24个tRNA基因、2个rRNA基因、2个控制区、1个轻链复制起点和比典型基因组多的13个间隔子。和经典鱼类的线粒体基因组相比, 褐斜鲽和满月沙鲽都多了tRNA-Cys和tRNA-Tyr两个tRNA, 冠鲽只多了tRNA-Cys, 且3个种类都多了一个控制区, 但褐斜鲽与满月沙鲽的基因排列顺序相同。褐斜鲽线粒体基因的重排导致不同位置的6个tRNA形成了六连体基因簇“tRNA-Cys₁-Tyr₁- Ser1-Lys-Arg-Ser2”, “ND5-ND6-Glu-Cytb-Thr”则位于六连体之后, 但这11个基因相对的排序没有发生变化。采用双复制随机丢失模型(double replications and random loss, DRRL)对褐斜鲽基因的重排现象进行分析, 认为该鱼类基因数量、排列顺序以及比典型基因组多13个间隔子等特征为该模型提供了新的依据。     

Integration of biochemical, histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary, U.K

The aim of this study was to examine whether a combination of biochemical, histopathological and toxicogenomic data could be used as a valuable tool for the assessment of biological risk associated with pollutants within the Tamar River and Estuary, S.W. England, U.K. Accordingly, biochemical and histopathological biomarkers (protein carbonyls, lipofuscin, neutral lipids, lysosomal stability [N-acetyl-β-hexosaminidase and neutral red], lysosomal volume, ferric reducing antioxidant power [FRAP] and malonaldehyde [MDA]) and gene expression profiles were assessed in 5 sites from the Tamar River and Estuary (Neal Point, Town Quay, Wilcove, Cremyll Ferry and Whitsand; and a reference site, Trebarwith Strand, N. Cornwall). PAHs were measured in mussel tissue and sediment and metals were measured in mussel tissue only. Data from the biomarkers was integrated into a Mussel Expert System (MES) model to produce a simple assessment of mussel stress. Clear gradients of mussel toxicity were identified by the biomarkers (with the exception of neutral lipids) with the highest impacted animals found furthest up the Tamar, whilst the MES was unable to identify a gradient of effect. Gene expression profiles also indicated a gradient of stress with the greatest number of significantly up- or down- regulated genes found at the uppermost 2 sites. The MES did, however, determine that mussels from all sites, except the reference site, were highly stressed; a conclusion that could not be inferred from the biomarker data alone. It is concluded that the MES is a valuable tool that permits integration and interpretation of complex sets of biomarker data by identifying the biological meaning of biomarker changes.     

低盐胁迫下松江鲈aquaporin基因的表达变化规律

为了探究aquaporin基因家族成员在松江鲈(Trachidermus fasciatus)应对低盐胁迫过程中的调节作用,本研究选取7个Aquaporin基因（aqp1、aqp4、aqp7、aqp8、aqp10、aqp11和aqp12）为目标基因,利用实时荧光定量PCR技术检测了鳃、肠、肾和肝组织中7个基因在两种低盐胁迫处理下不同时间点(0 h、12 h、24 h和48 h)的表达水平。在两种低盐胁迫处理下,aqp4在松江鲈鳃、肝和肠组织中无表达,aqp7在鳃、肝组织中无表达。此外,其他aqp基因在四个组织中的表达量变化趋势具有显著差异。除肾组织中aqp12和肠组织中的aqp7、aqp8和aqp10之外,特定组织中aquaporin基因在两种低盐胁迫下呈现相似的表达量变化趋势。本研究通过比较分析了aquaporin基因在松江鲈应对不同低盐胁迫时表达变化规律的异同,相关结果为探讨这一家族成员在鱼类应激调节过程中的作用及洄游性鱼类适应盐度变化的分子调控机制提供了理论依据。