EDCs对鱼类种群与生物多样性的影响

环境内分泌干扰物( Environmental endocrine-disrupting chemicals,EDCs)具有存在的广泛性和生物的难降解性,能够对自然界生物包括人类产生普遍的内分泌干扰效应.EDCs暴露可以引起鱼类内源性激素代谢过程的异常及其他功能异常,改变基因表达,诱导基因突变,引起生殖缺陷、发育异常、生长畸形、免疫功能下降及后代性别比例失衡,影响其存活率及后代繁殖数量,从而导致鱼类种群数量减少.通过降低对环境抑制因素的适应性和破坏生物网结构的稳定性,EDCs能够广泛地削弱生物的进化潜能,影响局部生态系统的功能,从而导致生物多样性丢失.针对EDCs的相关研究对人类社会的可持续发展有重要意义.     

Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.     

鱼类白介素10的研究进展

白介素10(Interleukin 10, IL-10)是能够参与机体免疫的一种多功能细胞因子,由单核细胞、巨噬细胞等多种免疫细胞分泌,在抗肿瘤、抗感染、免疫性疾病等多种疾病的发生发展过程中发挥重要作用。有多种硬骨鱼IL-10被克隆表达,也被证实具有免疫调节作用,目前也有不少关于鱼类IL-10的生物活性、作用机制、调节机制的研究。本文根据已有报道,从鱼类IL-10的基因结构、转录表达、来源与进化、生物学活性及功能、鱼类IL-10受体及信号通路等五个方面进行了综述,为鱼类IL-10的研究与应用提供依据。     

一种(鱼师)鱼诺卡氏菌酪氨酸蛋白磷酸酶基因的克隆及功能初步研究

這鱼诺卡氏菌是鱼类诺卡氏菌病的主要病原,可导致鱼类慢性系统性肉芽肿疾病.這鱼诺卡氏菌全基因组序列分析发现了一个酪氨酸蛋白磷酸酶(protein tyrosine phosphtase,PTP)基因,生物信息学分析显示该基因很可能编码一个靶向定位于宿主细胞线粒体的分泌蛋白.本实验对這鱼诺卡氏菌PTP进行了基因克隆、分泌蛋白鉴定、亚细胞定位、过表达和线粒体膜电位检测,结果显示,在這鱼诺卡氏菌胞外产物中质谱鉴定到了PTP肽段,证实其为分泌蛋白.亚细胞定位研究观察到PTP-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明這鱼诺卡氏菌PTP蛋白并未靶向定位于线粒体.亚细胞定位和过表达研究都显示PTP蛋白在FHM细胞中表达后,细胞核出现固缩浓染、凋亡小体等明显的细胞凋亡特征.通过线粒体膜电位检测表明,在pcDNA-PTP转染后48 h,线粒体跨膜电位被明显破坏,说明這鱼诺卡氏菌PTP很可能是一种可诱导细胞凋亡的细菌蛋白.通过对這鱼诺卡氏菌PTP开展基因克隆和功能初步研究,为进一步揭示该基因的功能和深入了解這鱼诺卡氏菌的分子致病机理奠定了基础.     

鱼类性别决定的研究进展

有关鱼类性别决定的研究主要集中在温度、性激素、芳香化酶以及随机重复序列、核受体基因等对性别分化的调控方面。由于鱼类所处分类地位较低 ,生活环境千差万别 ,鱼类性别决定没有一个普遍的模式 ,目前研究的鱼类又各不相同 ,因此象哺乳动物那样的性别决定级联模式还没能阐述。本综述旨在阐述近几年有关鱼类性别决定机制方面的研究动态和进展 ,为系统研究鱼类性别决定机制提供参考     

用RNA/DNA比率评定鲤的生长及其配合饲料的营养价值

投饲不同动物蛋白源，不同蛋白质含量的配合饲料养殖鲤鱼种，在环境条件和投饲率相同的情况下，测定鱼体的增重率、肌肉、肝脏中脱氧核糖核酸（ＤＮＡ）和核糖核酸（ＲＮＡ）的含量。结果表明：Ａ、Ｂ、Ｃ三组饲料蛋白质含量分别为１８．１７％、１７．８６％、３１．５６％时，其个体平均增重率分别为１０６．８７％、９４．６５％、１２９．４８％；平均饲料系数分别为２．０４、２．３０、１．５２时，ＲＮＡ／ＤＮＡ平均比率分别     

盐藻钙依赖蛋白激酶基因DsCDPK的表达分析

以盐藻总RNA为模板,利用RT-PCR技术扩增了盐藻DsCDPK基因的cDNA序列,将其克隆到pMD^TM19-T simple载体上,经测序获得的克隆片段全长1650 bp,与已发表的盐藻DsCDPK(GenBank:JQ964113)的编码区序列同源性达100%。将DsCDPK基因的开放阅读框与质粒pET-32a(+)连接,构建原核表达载体pET-32a-DsCDPK。将该重组质粒转化到大肠杆菌BL21中,经IPTG诱导融合,蛋白在大肠杆菌BL21中得到成功表达。通过SDS-PAGE检测发现,融合蛋白为部分可溶性表达,将上清蛋白经过His柱纯化后获得了纯度较高的可溶性融合蛋白,Western杂交检测显示,融合蛋白能被抗His单克隆抗体特异性识别,初步证明该融合蛋白就是带有His标签的DsCDPK蛋白。采用实时荧光定量PCR方法分析了高盐胁迫下DsCDPK基因的表达模式。试验结果表明,盐藻DsCDPK基因为盐胁迫上调基因,在高盐(3.0 mol/L NaCl)胁迫下,DsCDPK基因表达量显著增加,盐胁迫1 h时表达量达到最高,为正常生长状况(1.0 mol/L NaCl)下的3倍,差异达到极显著水平(P<0.01)。该研究成果为进一步阐明盐藻钙依赖蛋白激酶基因的功能及作用机制奠定了基础。     

Complete sequencing of Tunisian redspotted grouper nervous necrosis virus betanodavirus capsid gene and RNA‐dependent RNA polymerase gene

Finfish nodaviruses (betanodaviruses) can cause highly destructive infections in numerous species of farmed marine fish larvae and juveniles worldwide. The betanodavirus genome consists of two single‐stranded positive‐sense RNA molecules (RNA1 and RNA2). The virus can be classified into four genotypes based on the partial sequences of the coat protein (CP) gene (T2 and T4 regions). Currently, genomic sequence information for RNA1 regions of RNA2 outside of T2 and T4 is less well documented. This study reports on the characterization of the full RNA2 sequence of a Tunisian betanodavirus with a length of 1433 nt, containing a 339 amino acid open‐reading frame encoding the CP, and typing to the redspotted grouper nervous necrosis virus Ia genotype following phylogenetic analysis. The homology of the capsid protein to other betanodaviruses or alphanodaviruses was compared. In addition, a full length RNA1 sequence of 3104 nt encoding a 982 amino acid RNA‐dependent RNA polymerase was obtained.     

Development of a new antibody for detecting natural killer enhancing factor (NKEF)-like protein in infected salmonids

The main cellular responses of innate immunity are phagocytic activity and the respiratory burst, which produces a high amount of reactive oxygen species. Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family that has an antioxidant function and enhances cytotoxic cell activity. This molecule may play a key role in macrophage and cytotoxic cell communication during the innate immune response of fish against pathogens. In fish, the NKEF gene has been characterized in some species as showing an up-regulation in infected fish, suggesting a trigger effect upon NK-like cells. To detect and localize this molecule in salmonids at protein level, a monospecific polyclonal antibody was generated. A probable NKEF-like protein epitope region was identified and characterized using bioinformatic tools, and the sequence was chemically synthesized using Fmoc strategy, analysed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was immunized and antibodies from ascitic fluid were obtained. The resulting antibody is a versatile tool for detecting NKEF by different immune techniques such as ELISA, Western blotting and immunohistochemistry. Analysis of NKEF-like protein is a useful method for characterizing immune properties of this molecule in fish during response to pathogens.     

金钱鱼孕激素受体基因的克隆、鉴定与表达研究

核受体(Pgr),隶属于核受体超家族,由核受体亚家族3C组成员3基因编码.孕酮通过结合Pg r介导其在脊椎动物繁殖过程中的生理学功能,因此,研究金钱鱼核受体基因(pg r基因)在不同组织及不同发育时期性腺中的表达模式,有助于为金钱鱼的繁殖提供理论依据.笔者克隆金钱鱼的pg r基因,用Megalign软件比较Pgr氨基酸...     

褐牙鲆中miRNAs与甲状腺激素受体TRβ的相互作用研究

甲状腺激素通过与其受体结合对褐牙鲆变态起重要的调控作用,而miRNAs通过结合靶基因3′-UTR在转录后水平调节靶基因的表达。为探讨miRNAs在褐牙鲆变态中的作用,通过生物信息学方法预测褐牙鲆TRβ基因3′-UTR存在的miRNAs结合位点,并利用3′-RACE方法克隆TRβ基因的3′-UTR序列,通过体外DNA重组技术构建应用于miRNAs靶向基因检测的双荧光素酶重组报告载体,利用细胞转染和双荧光素酶报告技术分析多个miRNAs与TRβ基因的作用关系。研究结果显示,克隆得到了一段长为437 bp的褐牙鲆TRβ基因的3′-UTR序列,发现3′-UTR序列上存在pol-miR-125a、pol-miR-214、pol-miR-460-5p、pol-miR-138和pol-miR-125a*的结合位点,成功构建了双荧光素酶重组报告载体,命名为pmirGLO-TRβ-3′-UTR。双荧光素酶报告分析结果表明,pol-miR-125a、pol-miR-214、pol-miR-460-5p和pol-miR-138均为作用于褐牙鲆TRβ的靶向miRNAs,而pol-miR-125a*对TRβ基因无直接的调节作用。研究结果将为后续深入研究miRNAs与TRβ在褐牙鲆变态发育中的功能及其作用机制提供基础。     

养殖大黄鱼一种黏孢子虫病的组织病理学及检测方法初探

运用组织病理切片、电镜观察及PCR扩增等方法对2017年在舟山采集到的临床表现"白鳃"症状的发病大黄鱼(Larimichthyscrocea)开展了病原学及快速检测方法的初步研究。组织病理观察显示,患病鱼的肝、脾、肾等内脏组织发生严重的病理变化,尤其是组织内红细胞发生明显的退行性变化,同时血液中红细胞数量显著减少。在病鱼组织的电镜超薄切片中可观察到直径约300～600 nm的孢子虫样结构。提取病鱼内脏组织总基因组DNA样本,采用1对针对寄生原虫的通用引物进行SSUrDNA的PCR扩增,最终获得大小为1471bp的特异性条带,经测序比对发现,该条带与GenBank中1种黏孢子虫Sinuolinea sp.的序列同源性最高,达89%。根据获得的SSU rDNA序列,建立了适用于该病临床检测的巢式PCR方法,最小灵敏度可达0.5 pg。研究表明,引起此次网箱养殖大黄鱼"白鳃"症状并导致鱼类大量死亡的是一类寄生性黏孢子虫。     

Myosin expression levels in trout muscle: a new method for monitoring specific growth rates for rainbow trout Oncorhynchus mykiss (Walbaum) on varied planes of nutrition

Fish growth is manifested by a number of measurable physical changes. We have developed a sensitive method for monitoring the growth rate of fish fed at four different planes of nutrition. This technique consists in measuring expression levels of myosin RNA isolated from the muscle of experimental animals. Using PCR (polymerase chain reaction) primers, and a fluorescence-labelled single-stranded DNA probe that hybridizes specifically to a region within the myosin mRNA of rainbow trout Oncorhynchus mykiss (Walbaum), we were able to detect differences in the relative level of myosin expression between groups of fish. This method also allows for the determination of absolute expression levels when reactions are performed with standards consisting of known levels of in vitro-transcribed myosin RNA. With the proper equipment, this novel procedure can be performed rapidly on large numbers of individuals, and with the procurement of non-invasive muscle biopsies the same experimental animal could theoretically be sampled multiple times throughout the course of the study. This new method could be used to measure differences in muscle synthesis in fish associated with various nutrient intake levels, environmental parameters, life-history stages and health status.     

Characterization and distribution of Staphylococcus sp. implicated for improvement of fish sauce odor

ABSTRACT:   The two Staphylococcus strains that had been isolated from fish sauce mush (moromi) made from frigate mackerel in Japan and proved to improve fish sauce odor, were examined for their taxonomic positions. The sequence analysis based on 16S rRNA and rpoB showed that the two strains, R4Nu and R5G, had an identical sequence with sequence identities of 99.5% and 99.0% to the above two genes from the closest species of S. nepalensis , respectively. A DNA hybridization test of the two strains showed more than 80% DNA similarity with S. nepalensis , thus confirming the above-mentioned species identification. Polymerase chain reaction primers specific to the strain isolated from fish sauce mush were designed from rpoB and examined for the distribution of this species to various fish sauces made in Asian countries as well as to fish sauce starter (malt) made from soy beans and barley in Toyama Prefecture, Japan. The amplified DNA fragment bearing the S. nepalensis gene was detected in the enriched culture of the malt, although no positive reaction was shown with fish sauce samples. These results suggest that S. nepalensis indebted to improve fish sauce odor was originated from the fish sauce starter malt.     

草鱼LPL基因的表达及饥饿和再投喂对其影响

获得了草鱼脂蛋白脂酶(lipoprotein lipase, LPL)部分cDNA序列（GenBank注册号为FJ612596）,并进行了序列同源性分析;采用半定量反转录聚合酶链式反应（SQ-RT-PCR）方法,检测了LPL基因在草鱼不同组织和不同发育阶段脂肪细胞中的表达状况;研究了饥饿和再投喂对草鱼肝胰脏LPL基因表达的影响。结果显示,所获得的草鱼LPL部分cDNA序列长度为360 bp,与其它物种的同源性为70%~89%; LPL基因在肝胰脏、肌肉、心脏、鳃、腹腔脂肪组织中均表达,其中在腹腔脂肪组织中表达丰度最高,在鳃中表达丰度最低;在成熟脂肪细胞中的表达丰度显著高于基质脉管组分（stromal vascular fraction,SVF）细胞;草鱼LPL基因表达水平在饥饿48 h后显著升高,再次投喂后12 h,回复到0 h的表达水平。研究表明,草鱼LPL在不同组织及脂肪细胞分化的起始和终末阶段均有表达,显示其在草鱼不同组织和脂肪细胞分化中具有重要作用,同时,其表达水平受营养状况的调控。论文首次克隆得到草鱼LPL基因部分cDNA序列,并对其进行了表达分析,研究结果可为LPL在鱼类脂质代谢中的作用研究提供参考和依据。     

MHC基因在鱼类遗传育种中的研究与应用

主要组织相容性复合体（MHC,Major histoeompatibility complex）是最具多态性的免疫分子。其多态性主要取决于群体内MHC基因的大量等位基因及等位基因间高度的序列变异。等位基因的序列变异主要发生在抗原肽结合区（peptide binding region,PBR）,这决定了不同基因型个体的免疫力差异。鱼类受多倍化影响,MHC基因的多态性还表现在同一基因存在多个基因座位（多拷贝）。MHC的高度多态性及与机体免疫力的相关性,使其在鱼类遗传进化、抗病育种等方面倍受关注。本文介绍了有关鱼类MHC基因的结构、功能及其在遗传育种中的应用。     

鳀科(Engraulidae)鱼类DNA条形码电子芯片研究

本研究将基于线粒体COⅠ部分序列的DNA条形码和DNA芯片技术相结合,以鳀科11属30种鱼类为研究对象,在比对分析其DNA条形码序列的基础上,利用软件Oligo Array 2.1筛选探针,经OligoCalc优化探针,去除易形成发夹(Hairpin)、茎环(Stem-loop)及自身二聚体结构(Homodimers)的探针,再利用Oligo heat map对探针与靶标序列进行虚拟杂交,共有14个物种的24条探针能与靶标序列特异性结合.利用DNA条形码芯片技术能将14个物种鉴定到种,虽然物种识别能力仅占总物种数的46.7％,但鉴定准确率可达100％.因此,基于COⅠ基因的DNA芯片技术对鳀科鱼类物种鉴定有一定的实用价值,但该技术对物种的识别能力尚有较大提升空间,通过筛选和优化得到高质量的分子探针则是突破该项技术的关键.     

Rapid detection and identification of viral and bacterial fish pathogens using a DNA array-based multiplex assay

Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study.