鱼类microRNA研究进展

microRNA是一种大小为18~25个核苷酸的非编码RNA,调控靶基因的转录后表达,广泛参与多种生理过程。本研究简要综述了microRNA生物学特性、转录后调控机制,详细讨论了第二代测序技术的相关应用、microRNA在鱼类中的表达模式、功能研究,包括渗透压调节、生殖、发育、生长、免疫、代谢等。现有的研究表明,miR-200和miR-30具有调控血浆离子浓度和渗透压平衡的功能;Dicer1是胚胎发育所必需的,在原肠胚时期,miR-20a调节头、眼、脊柱、体节的形态发生,miR-92调控内胚层的形成,miR-10影响体轴形成,miR-219影响头部和尾部细胞的凋亡;miR-122、miR-30、miR-145分别影响肝细胞成熟、肝胆管和肠道发育,miR-138和miR-143调控心脏发育,miR-126、miR-150、miR-451调控血小板、血细胞的发育和成熟,miR-10和miR-126影响血管生成,miR-200、miR-183分别影响嗅觉、听觉系统的发育,多个microRNA参与视网膜、肌肉、鱼鳍、肝脏等器官的再生;miR-133a/b、miR-206抑制肌肉细胞分裂,miR-214、miR-499、miR-199、miR-3906调控快肌纤维和慢肌纤维的生成;miR-430促进原始生殖细胞的生成;多个microRNA参与到禁食和再投喂调控的分解代谢和合成代谢平衡;此外,microRNA通过对免疫因子的调控参与到先天免疫、低氧适应、体色调控、应激反应、肌间刺形成等生理过程中。本研究通过对这些结果进行总结和讨论分析,将有助于对鱼类microRNA研究领域现状的了解和把握,促进鱼类非编码RNA研究的深入。     

硬骨鱼免疫相关microRNA研究进展

MicroRNA (miRNA)是一类长度为18～25 nt的非编码RNA,在硬骨鱼免疫应答中起重要作用。近年来,借助高通量测序与荧光定量PCR (RT-PCR)技术,筛选到大量免疫相关的差异表达miRNA (DE miRNA)。通过比较多种硬骨鱼在不同病毒/细菌感染条件的miRNA表达谱,筛选出相同DE miRNA,并认为筛选出的DE miRNA是潜在参与调节宿主免疫应答的进化上保守的候选DE miRNA。据此,我们在病毒感染表达谱中筛选到10个进化保守的DE miRNA,细菌感染表达谱中筛选到8个保守的DE miRNA。此外,miRNA的功能研究也取得较大进展,宿主miRNA不仅可直接抑制病毒的复制,同时参与调控干扰素反应、炎症、细胞凋亡等过程。本文简述了硬骨鱼免疫相关miRNA的研究进展,以期为硬骨鱼疾病预防与治疗提供新思路。     

首次报道病毒miRNA调控对虾抗病毒

<正>病毒,尤其是DNA病毒,会产生自身的microRNA(miRNA)来调控宿主和病毒基因的表达。由于其在病毒-宿主相互作用中的重要作用,近年来病毒miRNA已引起了广泛的关注与研究,然而人们对无脊椎动物病毒miRNA的了解却十分有限。大多数对病毒miRNA的研究是在细胞系中进行的,但是病毒miRNA在细胞系中发挥的作用可能与其在宿主体内的作用差异巨大。浙江大学生命科学学院章晓波教授课题组首次对对虾体内的白斑综合症病毒(WSSV)编码的miRNA进行了表征分析,     

长牡蛎性腺中调控型非编码RNA的生物信息学

本研究以日照海域同一家系的2龄长牡蛎性腺为研究对象,通过small RNA-seq和RNA-seq技术筛选和鉴定出大量的非编码微小RNA(mi RNA)、长链非编码RNA(lnc RNA)和环状RNA(circRNA),并对其进行了生物学特征分析。结果显示,以斑马鱼为参考序列,获得25～30个已知miRNA成熟体和51～63个已知miRNA前体,预测到53～71个新miRNA成熟体和53～77个新miRNA前体;长牡蛎miRNA长度分布为18～26个核苷酸(nt),其中分布在20～22 nt长度的miRNA数量最多,且miRNA首位碱基多为U。测序分析获得2 302～2 349个注释lncRNA转录本,预测到20 083～24 114个新lncRNA转录本,其中基因间型lncRNA(lincRNA)、内含子lncRNA(intronic lncRNA)、反义lncRNA(anti-sense lncRNA)分别占29.0%、62.1%和8.9%;长牡蛎lncRNA的基因组特征与其他真核生物的lncRNA基因组特征相似,与mRNA相比,外显子(exon)个数少,转录本长度较短,表达水平低。测序分析共获得383个circRNA,其中平均88.54%来源于exon,平均4.51%来源于intronic,平均6.95%来源于intergenic,且鉴定出内源性circRNA潜在大量的miRNA结合位点。研究结果为后续研究长牡蛎调控型ncRNA的表达规律和生物学功能奠定了基础。     

环状RNA在动物肌肉发育中的研究进展

环状RNA是一种非编码RNA,不具有游离的5′端和3′端结构,而是通过反向剪接形成闭合结构,稳定性极高。已有研究证明,环状RNA参与骨骼肌生长发育;可以作为竞争性RNA调节miRNA的活性和水平;可作为转录本剪接;具有编码潜能。环状RNA可以与RNA结合蛋白结合发挥作用,在动物肌肉发育中起着重要作用。阐述了环状的生物学特性功能,重点阐述环状RNA在动物肌肉发育中的研究进展。     

摄食不同种类饵料的鲤肝脏组织miRNA表达谱分析及验证

MicroRNA(miRNAs)是一类长度为18-25nt的内源性非编码单链小RNA,参与细胞增殖与凋亡、免疫、脂肪代谢等过程。本试验以三组投喂不同类型饵料的鲤(Cyprinus carpio)肝脏为研究材料,通过HiSeq 2500深度测序技术和生物信息学分析miRNA,鉴定出235个已知miRNAs。三种饵料组共表达的差异miRNAs有13个,随机挑选5个miRNA并采用qPCR进行验证,表达趋势在qRT-PCR结果和RNA测序结果中高度相似;对13个共表达的差异表达miRNAs进行靶基因预测,共预测到靶基因530个。对预测靶基因进行KEGG通路分析,结果显示参与2-羰基羧酸代谢,糖基磷脂酰肌醇(GPI)-锚定生物合成,柠檬酸循环(TCA循环)等通路的调节。本研究首次了解鲤摄食不同种类饵料的miRNAs表达特征,可以为深入了解miRNA对鲤摄食不同种类饵料过程的调控作用奠定基础。     

鱼类microRNA在先天免疫中的研究进展

先天免疫是宿主识别病原及消除病原感染的第一道防线。模式识别受体是参与识别病原入侵的主要分子，主要包括Toll样受体、RIG-I样受体、NOD样受体和C型凝集素受体等。模式识别受体在识别病原相关分子模式后，激活机体的先天免疫信号通路，诱导炎症细胞因子和干扰素的产生，从而启动抵抗病原入侵的免疫应答。越来越多的证据表明，免疫应答的激活、维持和终止受到了严格的调节，使机体在保持一定免疫强度的同时避免产生过度的免疫反应。microRNA是一类长度为18～23 nt的微小非编码RNA，是鱼类先天免疫应答网络中的重要调控因子。近年来，microRNA在鱼类免疫学领域已开展了大量的研究，但缺乏对其进行及时地全面性的总结。本文综述了近年来miRNA在鱼类先天免疫反应中的研究进展，以期为鱼类的分子抗病育种及疾病防控研究提供一些思路。     

半滑舌鳎(Cynoglossus semilaevis) miR-223的表达特征及免疫应答分析

microRNA(miRNA)是一类内源性、长度约为22个核苷酸的非编码小单链RNA分子,由具有发夹结构的70-90个碱基的单链RNA前体经过Dicer酶加工而成.本研究使用荧光实时定量PCR (Quantitative Real-Time PCR, qRT-PCR)方法,研究了miRNA-223(miR-223)在半滑舌鳎(Cynoglossus semilaevis)各健康组织、鳗弧菌(Vibrio anguillarum)感染后各时间点的免疫组织以及不同病原类似物刺激后头肾细胞中的表达模式.结果显示,miR-223在半滑舌鳎各组织中均有表达,在头肾中表达量最高,在脑和血液中的表达量极低.鳗弧菌感染3组样品4种免疫组织,miR-223表达变化显著,感染后20 h内,鳗弧菌诱导miR-223上调表达.鳗弧菌感染后半滑舌鳎免疫组织miR-223表达变化规律显示,鳗弧菌感染2、6、12、24、48、72、96和168 h后,miR-223在肝、肠、脾、头肾4种组织中出现差异表达.其中,miR-223在半滑舌鳎肝、脾、头肾中表达上调,在肠中表达下调.用LPS、poly I:C、PGN、RGNNV感染半滑舌鳎头肾细胞后,发现经LPS、RGNNV诱导后miR-223上调表达,poly I:C、PGN诱导后miR-223下调表达.研究结果表明,miR-223参与了半滑舌鳎免疫应答过程.本研究结果有助于了解miRNA在半滑舌鳎对病原刺激免疫应答过程中的作用以及半滑舌鳎与病原相互作用中miRNA参与调控的机制.     

半滑舌鳎(Cynoglossus semilaevis) miR-223的表达特征及免疫应答分析

microRNA(miRNA)是一类内源性、长度约为22个核苷酸的非编码小单链RNA分子，由具有发夹结构的70?90个碱基的单链RNA前体经过Dicer酶加工而成。本研究使用荧光实时定量PCR (Quantitative Real-Time PCR, qRT-PCR)方法，研究了miRNA-223(miR-223)在半滑舌鳎(Cynoglossus semilaevis)各健康组织、鳗弧菌(Vibrio anguillarum)感染后各时间点的免疫组织以及不同病原类似物刺激后头肾细胞中的表达模式。结果显示，miR-223在半滑舌鳎各组织中均有表达，在头肾中表达量最高，在脑和血液中的表达量极低。鳗弧菌感染3组样品4种免疫组织，miR-223表达变化显著，感染后20 h内，鳗弧菌诱导miR-223上调表达。鳗弧菌感染后半滑舌鳎免疫组织miR-223表达变化规律显示，鳗弧菌感染2、6、12、24、48、72、96和168 h后，miR-223在肝、肠、脾、头肾4种组织中出现差异表达。其中，miR-223在半滑舌鳎肝、脾、头肾中表达上调，在肠中表达下调。用LPS、poly I:C、PGN、RGNNV感染半滑舌鳎头肾细胞后，发现经LPS、RGNNV诱导后miR-223上调表达，poly I:C、PGN诱导后miR-223下调表达。研究结果表明，miR-223参与了半滑舌鳎免疫应答过程。本研究结果有助于了解miRNA在半滑舌鳎对病原刺激免疫应答过程中的作用以及半滑舌鳎与病原相互作用中miRNA参与调控的机制。     

半滑舌鳎(Cynoglossus semilaevis)miR-223的表达特征及免疫应答分析

microRNA(mi RNA)是一类内源性、长度约为22个核苷酸的非编码小单链RNA分子,由具有发夹结构的70-90个碱基的单链RNA前体经过Dicer酶加工而成。本研究使用荧光实时定量PCR(Quantitative Real-Time PCR,q RT-PCR)方法,研究了mi RNA-223(mi R-223)在半滑舌鳎(Cynoglossus semilaevis)各健康组织、鳗弧菌(Vibrio anguillarum)感染后各时间点的免疫组织以及不同病原类似物刺激后头肾细胞中的表达模式。结果显示,mi R-223在半滑舌鳎各组织中均有表达,在头肾中表达量最高,在脑和血液中的表达量极低。鳗弧菌感染3组样品4种免疫组织,miR-223表达变化显著,感染后20 h内,鳗弧菌诱导miR-223上调表达。鳗弧菌感染后半滑舌鳎免疫组织mi R-223表达变化规律显示,鳗弧菌感染2、6、12、24、48、72、96和168 h后,miR-223在肝、肠、脾、头肾4种组织中出现差异表达。其中,miR-223在半滑舌鳎肝、脾、头肾中表达上调,在肠中表达下调。用LPS、poly I:C、PGN、RGNNV感染半滑舌鳎头肾细胞后,发现经LPS、RGNNV诱导后mi R-223上调表达,poly I:C、PGN诱导后miR-223下调表达。研究结果表明,mi R-223参与了半滑舌鳎免疫应答过程。本研究结果有助于了解miRNA在半滑舌鳎对病原刺激免疫应答过程中的作用以及半滑舌鳎与病原相互作用中miRNA参与调控的机制。     

The relationship between T3 production and energy balance in salmonids and other teleosts

Extrathyroidal T₄ 5′-monodeiodination, demonstrated in several teleost species, generates T₃ which binds more effectively than T₄ to putative nuclear receptors and is probably the active thyroid hormone. T₄ to T₃ conversion is sensitive to the physiological state and provides a pivotal regulatory link between the environment and thyroid hormone action. T₃ generation is enhanced in anabolic states (positive energy balance or conditions favoring somatic growth; food intake or treatment with androgens or growth hormone) and is suppressed in catabolic states (negative energy balance or conditions not favoring somatic growth; starvation, stress, or high estradiol levels associated with vitellogenesis). In fish, as in mammals, thyroidal status may be finely tuned to energy balance and through T₃ production regulate energy-demanding processes, which in fish include somatic growth, development and early gonadal maturation.     

Induced maturation and spawning: opportunities and applications for research on oogenesis

Broodstock management requires the ability to detect and regulate oocyte growth, acquisition of maturational competence, maturation of oocytes, and onset of ovarian atresia. Our research on temperate basses (genus Morone) has supported development of these capabilities. These investigations have revealed that accumulation of neutral lipid droplets and deposition of vitellogenin-derived yolk proteins in growing oocytes are independent processes with different sensitivities to changing day length and water temperature. In these fishes, completion of oocyte growth is marked by disappearance of vitellogenin from ovarian biopsy samples. Competence of females for induced spawning is predicted by the ability of biopsied follicles to initiate oocyte meiosis in vitro in response to insulin-like growth factor I. Cytoplasmic maturation of the oocytes is triggered by the maturation-inducing steroid hormone and can be monitored by evaluating degradation of the yolk proteins. Onset of ovarian atresia is indicated by the appearance of edema in the granulosa cell layer of biopsied follicles, and can be delayed for months by holding gravid females at abnormally low temperature (`cold banking'). These novel findings hold strong promise for application to other farmed fishes.     

Energy dynamics of subyearling Chinook salmon reveal the importance of piscivory to short‐term growth during early marine residence

Variation in prey quantity and quality can influence growth and survival of marine predators, including anadromous fish that migrate from freshwater systems. The objective of this study was to examine the energy dynamics of subyearling Chinook salmon (Oncorhynchus tshawytscha) following freshwater emigration. To address this objective, a population of Chinook salmon and their marine prey were repeatedly sampled from June to September over 2 years in coastal waters off Oregon and Washington. Subyearlings from the same population were also reared under laboratory conditions. Using a bioenergetics model evaluated in the laboratory, we found that growth rate variability in the field was associated more with differences in northern anchovy (Engraulis mordax) consumption and less with variation in diet energy density or ocean temperature. Highest growth rates (2.43–3.22% body weight/day) occurred in months when anchovy biomass peaked, and the timing of peak anchovy biomass varied by year. Our results support a general pattern among subyearling Chinook salmon occurring from Alaska to California that feeding rates contribute most to growth rate variability during early marine residence, although dominant prey types can differ seasonally, annually, or by ecosystem. In the northern California Current, faster growth appears to be associated with the availability of age‐0 anchovy. Identifying factors that influence the seasonal development of the prey field and regulate prey quantity and quality will improve understanding of salmon growth and survival during early marine residence.     

水产养殖鱼类生长性状研究进展

生长性状是水产养殖鱼类最重要的经济性状之一,对水产养殖业的发展意义重大。通过以不同的养殖鱼类为对象,大量的研究结果表明,鱼类生长主要受环境、基因,以及基因与环境相互作用的影响,具体为:(1)环境是生长性状调控的外因,其对生长的影响一般呈现出剂量效应的规律。温度、光照、营养等主要环境因子的过量和不足均可能对鱼类生长产生不利影响,因此,寻求最优条件是制定最佳养殖环境的终极目标,人为调控多种环境因子在现代水产养殖业中具有重大的应用潜力。(2)基因是生长性状调控的内因,其对生长的影响很大程度上表现出因果效应的关系。某些基因的单碱基核苷酸多样性、基因结构变异、染色体倍性变化,以及转基因等都表现出对鱼类生长产生统计显著性的影响。鉴于生长是多基因控制的复杂数量性状,寻找主效基因并在选育中加以利用是改良生长性状的重要基础。高通量测序技术在生长相关候选基因的筛选以及辅助分子育种方面展现出强大的优势。(3)基因与环境相互作用的影响主要来自基因型对不同环境条件的适应性,具有特异性和复杂性的特点,因此目前对其量化研究非常有限。但在制定大规模商业育种计划之前,考虑基因与环境相互作用具有重要意义。综上所述,充分理解环境、基因,以及基因与环境相互作用对水产养殖鱼类生长的影响能更好地对其生长性状加以利用,从而最大限度地节约养殖成本和发挥生态效益。     

益生菌在盐碱养殖水体中调控作用的研究进展

我国内陆、滨海地区有丰富且尚未有效开发利用的盐碱水资源，在盐碱水域进行水产养殖已成为其重要的开发利用途径之一，而盐碱水体的高pH、高盐、高碱特点严重限制了水产养殖对象的生长发育。益生菌具有净化水质、调节pH、降低氨氮和提高鱼类盐碱耐受能力等综合作用，是一种高效无污染的水质改良剂。合理使用益生菌可有效改良盐碱水体，提高水产养殖效益。文章综述了光合细菌、乳酸菌、芽孢杆菌、酵母菌等不同菌种制剂及其在适应、改良盐碱水体方面的研究进展。     

脂肪酸对鱼类免疫系统的影响及调控机制研究进展

替代脂肪源的开发和利用是解决鱼油短缺问题的必然选择。然而,随着替代水平的提高,鱼体常常表现免疫水平和抗病能力降低。鱼油替代的本质为脂肪酸替代,深入研究脂肪酸与鱼类免疫的关系显得尤为重要。本实验综述了脂肪酸对鱼类免疫性能的影响及调控机制。饱和脂肪酸会降低鱼类免疫力,而适量添加n-3长链多不饱和脂肪酸(LC-PUFA)、共轭亚油酸(CLA)或提高n-3/n-6多不饱和脂肪酸(PUFA)的比例有利于鱼体免疫力发挥;饲料中脂肪酸主要通过细胞膜结构、信号传导、类花生四烯酸、细胞因子和类固醇激素等途径对鱼类免疫进行调控。脂肪酸与鱼类的免疫性能具有高度相关性,而调控机制的研究尚有较大空间。未来研究应该侧重于以下几个方面:脂肪酸对免疫相关转录因子的调控机制;鱼类肠道脂肪酸组成改变与菌群结构和免疫性能之间的相关性;环境因子对鱼体脂肪代谢和免疫力的影响;非脂肪酸成分(矿物质、维生素)对鱼类脂肪酸代谢和免疫过程的调控机制。     

jnk3基因在金鱼和斑马鱼不同发育时期胚胎与成体不同组织中的分化表达

JNKs是丝裂原活化蛋白激酶(MAPK)家族重要成员,参与应激反应和动物体轴的构建。为了进一步了解JNK家族在动物发育中的功能,首次分离和克隆了金鱼jnk3基因,研究了jnk3在金鱼和斑马鱼组织特异性和胚胎发育特异性表达状况。金鱼jnk3基因cDNA总长1 794 bp,其中阅读框1 293 bp,编码430个氨基酸。对jnk3基因序列及其推导的氨基酸序列进行同源性分析,结果显示,jnk3基因在脊椎动物较为保守,金鱼jnk3基因核苷酸序列与斑马鱼、人的同源性分别为94.1%和80.7%,金鱼jnk3基因氨基酸序列与斑马鱼、人的同源性分别为99.7%和93.4%。jnk3基因在鱼类成体中的表达存在着显著的组织差异,在金鱼和斑马鱼脑和精巢组织的表达具专一性,此外在斑马鱼心脏中也检测到jnk3的表达。RT-PCR检测结果显示,在鱼类胚胎发育中,神经胚期开始检测到jnk3基因mRNA表达,从神经胚到心跳期胚胎,jnk3基因表达水平逐渐增高,此后直至出膜一直保持较高的表达水平。研究表明,jnk3基因可能在鱼类后期的胚胎发育和成体脑、精巢与心脏发育中具有重要作用。     

Growth potential and habitat requirements of endangered age‐0 pallid sturgeon (Scaphirhynchus albus) in the Missouri River,USA, determined using a individual‐based model framework

An individual‐based model framework was used to evaluate growth potential of the federally endangered pallid sturgeon (Scaphirhynchus albus) in the Missouri River. The model, developed for age‐0 sturgeon, combines information on functional feeding response, bioenergetics and swimming ability to regulate consumption and growth within a virtual foraging arena. Empirical data on water temperature, water velocity and prey density were obtained from three sites in the Missouri River and used as inputs in the model to evaluate hypotheses concerning factors affecting pallid sturgeon growth. The model was also used to evaluate the impacts of environmental heterogeneity and water velocity on individual growth variability, foraging success and dispersal ability. Growth was simulated for a period of 100 days using 100 individuals (first feeding; 19 mm and 0.035 g) per scenario. Higher growth was shown to occur at sites where high densities of Ephemeroptera and Chironomidae larvae occurred throughout the growing season. Highly heterogeneous habitats (i.e., wide range of environmental conditions) and moderate water velocities (0.3 m/s) were also found to positively affect growth rates. The model developed here provides an important management and conservation tool for evaluating growth hypotheses and(or) identifying habitats in the Missouri River that are favourable to age‐0 pallid sturgeon growth.     

A framework for the study of macronutrient intake in fish

A good understanding of the capabilities of commercially reared fish species to regulate intake of specific macronutrients has potential economic, welfare and environmental benefits. We present a conceptual and experimental framework for studying macronutrient intake in fish. This ‘geometric’ approach addresses the multidimensional and interactive nature of nutrition. It was developed from work on insect herbivores and has successfully been applied to mammals and birds. The various components of the framework are introduced in simple outlines, and key experimental designs are described for assessing whether or not fish specifically regulate their intake of macronutrients, how they balance over-ingesting some nutrients against undereating others when provided with suboptimal diets, and how they regulate growth post-ingestively.