花鲈ncc、nkcc基因在海、淡水适应中鳃组织的表达与定位分析

为探究ncc、nkcc基因在花鲈渗透调节中发挥的作用,实验通过全基因组鉴定、多重序列比对、系统进化树构建以及蛋白结构预测对花鲈ncc进行了鉴定及序列分析,利用实时荧光定量PCR (qRT-PCR)检测ncc和nkcc在海水、淡水花鲈鳃组织中的表达水平,利用原位杂交技术确定ncc2和nkcc1a在海水及淡水花鲈鳃中的表达位置。结果显示,从花鲈中鉴定出2个ncc基因,即ncc1和ncc2,其编码序列(CDS)长度分别为2 691和3 120bp,编码896和1 039个氨基酸,在进化上具有保守性。ncc2在淡水花鲈鳃组织中的表达量显著高于海水,而nkcc1a在海水花鲈鳃组织中的表达量显著高于淡水,ncc1、nkcc1b、nkcc2在海淡水中的表达量则无显著差异。淡水适应过程中花鲈鳃组织中的ncc2的表达量逐渐上调,而nkcc1a的表达量逐渐下调;海水适应过程则呈现相反的表达趋势。此外,原位杂交结果显示,ncc2和nkcc1a基因分别位于淡水与海水中鳃组织的相邻鳃小片间的鳃丝上皮。以上结果表明,ncc2和nkcc1a基因分别编码淡水及海水花鲈鳃中重要的Na⁺及Cl     

绿色荧光蛋白基因向花鲈胚胎的转移及其表达

通过显微注射的方法,将绿色荧光蛋白(Green fluorescent protein,GFP)基因注射入花鲈(Lateolabrax japonicus)单细胞期受精卵动物极细胞质内。初步研究了显微注射后的花鲈胚胎的存活状况。在荧光显微镜下观察到了GFP的表达,多数胚胎表现为嵌合性表达。利用PCR技术,从花鲈尾芽期胚胎DNA中扩增出了特异片段,大小为750bp,表明显微注射胚胎中整合了外源GFP基因。     

花鲈irak4基因cDNA的克隆与表达分析

该研究基于Illumina MiSeq高通量测序技术,对周期性缺氧应激下花鲈(Lateolabrax maculatus)肠道菌群结构的变化进行分析,为研究其幼鱼肠道菌群对环境缺氧的适应机制提供参考依据。结果显示,周期性缺氧导致花鲈肠道菌群多样性和丰富度显著增加(P<0.05),群落结构复杂化,缺氧组和常氧组群落组成存在较大差异。缺氧组肠道菌群分类操作单元相比常氧组显著增加(P<0.05),其特有操作分类单元 (operational taxonomic unit, OTU)数达1 003个。门分类水平上,变形菌门、厚壁菌门和拟杆菌门为2组肠道菌群的主要组成菌门。与常氧组相比,缺氧组变形菌门相对丰度显著降低(P<0.05),而拟杆菌门相对丰度显著升高(P<0.05);纲水平上,缺氧组α变形菌纲和芽孢杆菌纲相对丰度显著降低(P<0.05),梭菌纲、γ变形菌纲和拟杆菌纲相对丰度显著升高(P<0.05)。此外,周期性缺氧应激还引起花鲈肠道内厌氧绳菌科、毛螺菌科、瘤胃菌科等厌氧或兼性厌氧菌和绿硫菌科等光合产氧菌类相对丰度升高。

     

花鲈性腺分化组织学及性别相关基因cyp11b和cyp19a1a的表达分析

以花鲈(Lateolabrax maculatus) 1~214 dph (day post hatching)的仔稚鱼、幼鱼以及18月龄的雌鱼和雄鱼为研究对象,研究了花鲈早期性腺发生、发育和分化情况;分析了性腺分化过程中性别相关基因(cyp11b和cyp19a1a)的表达及与性别之间的关系。结果显示,在30 dph [全长为(1.28± 0.10) cm],首次在中肾管前端的腹腔膜周围观察到原始生殖细胞(primordial germ cells, PGCs),说明30 dph前是花鲈胚后PGCs迁移至生殖嵴的关键时期;在55 dph [全长为(2.45±0.19) cm],观察到一对呈对称分布的原始性腺已经形成,说明花鲈幼鱼的原始性腺在30~55 dph (全长为1.28~2.45 cm)之间发生;55~180 dph时(全长为2.45~12.28 cm),原始性腺不断发育变大,并且一直处于未分化状态;180 dph后性腺开始分化;在195 dph [全长(14.54±1.54) cm]观察到精巢开始分化,卵巢于205 dph [全长为(15.86±0.94) cm]开始分化,且性腺的解剖学分化要早于细胞学分化;18月龄的花鲈幼鱼性腺发育到Ⅱ期。性别分化相关基因cyp19a1a在花鲈卵巢中的表达量高于同期精巢,说明其在卵巢的分化及维持中发挥更关键的作用,而cyp11b在18月龄幼鱼Ⅱ期精巢中的表达量显著高于同时期的卵巢及Ⅰ期精巢,说明其主要在精巢的分化及维持中扮演重要角色。本研究结果不仅可以丰富花鲈的繁殖生理学资料,也为其性别调控技术的研究提供了科学依据。     

花鲈白细胞介素8基因的克隆与序列分析

采用同源克隆和末端快速扩增(RACE)的方法,从经LPS刺激的花鲈(Lateolabrax japonicus)总RNA反转录产物中获得了803 bp的IL-8 cDNA序列。该序列包含159 bp的5′非编码区(UTR)和359 bp的3′非编码区(UTR)以及297 bp的开放阅读框(ORF),可编码99个氨基酸。序列的3′UTR含2个mRNA不稳定基序,在Poly A尾上游17 bp处有1个明确的Poly A加尾信号(AATAA)。预测的氨基酸结构含27个氨基酸组成的信号肽,将信号肽切除可产生72个氨基酸组成的成熟肽,预测成熟肽的分子量约为8.3 kD,等电点为7.85。花鲈IL-8序列中用于形成二硫键的4个半胱氨酸保守,前2个半胱氨酸被1个精氨酸分开,形成CXC结构。与其他鱼类的IL-8相似,花鲈IL-8序列中也缺乏哺乳类中存在的ELR基序,而含有ELH结构。进化分析显示,花鲈IL-8与从鱼类和哺乳类分离的IL-8有不同程度的同源性,与硬骨鱼黑鲷(Acanthopagrus schlegeli)和牙鲆(Paralichthys olivaceus)的进化关系最近,其次为软骨鱼类,与无颌类和哺乳类的分化较大;而哺乳类的ELR结构可能为IL-8在进化过程中特化而成的结构,以加强IL-8特异性的趋化能力。     

HACCP体系在花鲈网箱养殖中的应用与探讨

文章对HACCP在花鲈网箱养殖中的应用试验进行了总结与探讨,旨在为HACCP体系在花鲈网箱养殖中的应用与推广提供参考依据。     

中国沿海6个花鲈群体的形态差异分析

通过聚类分析、主成分分析和判别分析等多元分析方法,对东港、绥中、秦皇岛、青岛、舟山以及珠海等6个花鲈(Lateolabrax maculatus)地理群体的8个形态比例性状进行了研究。聚类分析和主成分分析表明,6个花鲈群体可被分为两支:来自黄海、渤海海域的东港、秦皇岛、绥中和青岛群体为一支(北方群体);东海海域的舟山群体和南海海域的珠海群体为另一支(南方群体)。南北群体间有一定程度的形态分化,和舟山群体相比,珠海群体和北方群体的亲缘关系更近。主成分分析获得的3个主成分方差贡献率分别为31.726%、27.744%和14.075%,累计贡献率为73.545%。利用8个变量构建的6个地理种群的判别公式,判别准确率在63.6%~84.4%之间,综合判别准确率72.7%。本研究结果为花鲈地理种群判别、种质资源评价以及良种选育等提供了基础资料。     

花鲈肿瘤坏死因子基因cDNA的克隆、分析与表达

采用同源克隆法,结合锚定PCR技术,获得了花鲈(Lateolahrax japonicus)TNFα基因cDNA的部分序列。BLLAST分析表明,该序列与其他鱼类的TNFα基因具有很高的相似性与同源性。同时利用RT-PCR技术,对该基因在鱼体内不同组织之间的表达差异进行了分析研究,结果表明,该基因在花鲈免疫器官头肾、脾脏、肝脏中的表达较强,而在脑中的表达较弱,在肌肉中几乎不表达。而且在经特异性病原刺激前、后的组织对比分析中,发现鱼体经LPS(1ipopolysaccharide)刺激后,目的基因在免疫器官中的表达明显增强。该基因的克隆与表达为进一步深入研究海水鱼类的抗逆机理以及指导鱼类的遗传选育和遗传改良都具有重要的理论意义。     

花鲈不同养殖群体的遗传结构分析

采集我国花鲈(Lateolabrax maculatus)主要养殖地区山东(青岛市和东营市)、浙江(宁波市)、福建(福鼎市、漳州市梅岭镇、漳州市桥东镇和漳州市东山县)、广东(珠海市斗门区)的86尾个体作为实验对象,结合已报道的花鲈野生群体的遗传研究背景,通过ddRAD简化基因组测序对以上花鲈养殖群体进行遗传结构分析。主成分分析(PCA)与遗传组分分析结果(最佳聚类K值为2)均显示,8个花鲈养殖群体与天津、烟台和文登的野生群体间遗传差异较小,表明我国花鲈养殖群体主要为黄、渤海种质来源。8个养殖群体间遗传分化分析结果显示,福建漳州梅岭镇和桥东镇的2个养殖群体与其他养殖群体间出现显著的遗传差异,表明所检测的养殖群体内部也出现了一定程度的分化。本研究结果可为我国从山东到广东(从北到南)的花鲈养殖群体遗传结构分析提供参考,为后续花鲈种质资源的保护、育种工作提供依据。     

Correction to: Molecular characterization,expression following cold stress,and functional characterization of YB-1 gene in the spotted sea bass (Lateolabrax maculatus)

Aquaculture International -     

花鲈ISG15基因cDNA序列的克隆及传染性脾肾坏死病毒胁迫下表达谱分析

为探究花鲈(Lateolabrax maculatus)干扰素刺激基因 15 (Interferon-stimulated gene 15, ISG15)在抵抗病毒过程的免疫应答反应, 本研究克隆、鉴定了花鲈 ISG15 基因(LmISG15), 并对其进行生物信息学分析, 获得了 LmISG15 在各组织中的表达特征及其在受到传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus, ISKNV)感染后的时空表达特征。结果显示, 该基因全长 1625 bp, ORF 为 480 bp, 编码 160 个氨基酸, 分子质量为 17.5 kD, 理论等电点为 9.41。LmISG15 的氨基酸序列较为保守, 与眼斑拟石首鱼(Sciaenops ocellatus)和条石鲷(Oplegnathus fasciatus)相似度分别为 74.07%和 71.88%。LmISG15 在花鲈 10 种组织中均有表达, 在心脏中表达量最高, 其次在脑、中肾和鳃中的表达量较高, 在肌肉中表达量最低。注射 ISKNV 后, 花鲈头肾组织中病毒拷贝数升高, 同时 LmISG15 在花鲈肝脏、头肾和鳃中的表达量显著上调(P＜0.05)。研究表明, LmISG15 参与了抵抗病毒刺激的免疫应答过程。     

Outbreak of hirame rhabdovirus infection in cultured spotted sea bass Lateolabrax maculatus on the western coast of Korea

In this study, we determined the cause of a disease outbreak in spotted sea bass, Lateolabrax maculatus reared in culture cages on the western coast of Korea in 2013. The major signs in the diseased fish exhibited were haemorrhaging on the membranes of the abdomen, gastrointestinal organs and opercular gills, as well as an enlarged spleen. No external morphological signs of infection were visible, except for a darkening in colour. No parasites or pathological bacteria were isolated from the diseased fish; however, epithelioma papulosum cyprini (EPC) cells inoculated with tissue homogenates from the diseased fish showed cytopathic effects (CPEs). Virus particles in the EPC cells were bullet‐shaped, 185–225 nm long and 70–80 nm wide, characteristic of Rhabdoviridae. Polymerase chain reaction analyses of homogenized tissues from the diseased fish and supernatants of cell cultures with CPEs indicated specific, 553‐bp‐long fragments corresponding to the matrix protein gene of the hirame rhabdovirus (HIRRV). Phylogenetically, the HIRRV phosphoprotein gene of spotted sea bass was more closely related to phosphoproteins from Chinese and Polish HIRRV strains than from other Korean strains. To our knowledge, this is the first report of HIRRV infection in cultured spotted sea bass.     

Growth trials for polyculture of hatchery-reared juvenile spotted babylon (Babylonia areolata) and sea bass (Lates calcarifer) in a flow-through sea water system

Juvenile spotted babylon (Babylonia areolata) and sea bass (Lates calcarifer) were cultured in 11.5‐m³ indoor rearing tanks supplied with flow‐through of ambient natural sea water over a 120‐day experiment. Each species, stocked at the following densities for the following treatments, was tested with three replicates per treatment: 5000 B. areolata per tank (Treatment 1); 200 L. calcarifer per tank (Treatment 2); and 5000 B. areolata plus 200 L. calcarifer per tank (Treatment 3),. The average growth (length and weight), feed conversion ratio and total production of spotted babylon and sea bass from Treatment 3 were not significantly different from those of Treatment 1 and 2 (P > 0.05). Average survival rates for both spotted babylon and sea bass exceeded 95% for all treatments.     

Characterization of mevalonate metabolism in the sea bass (Dicentrarchus labrax L) liver

The activities of mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase, were examined in sea bass (Dicentrarchus labrax L) liver. The activities of the three enzymes were studiedin vitro in relation to the influence of protein content, time of incubation, pH, temperature, mevalonate, ATP and Mg⁺⁺ concentration. Protein content in the assay medium affected the three enzymes differently. Mevalonate kinase, mevalonate 5-phosphate kinase, and mevalonate 5-pyrophosphate decarboxylase activities were linear up to 0.2, 0.4 and 0.8 mg protein, respectively. With respect to the time course studies, the enzymes also behaved differently. Mevalonate kinase activity increased over forty minutes, reaching a plateau thereafter, while mevalonate 5-phosphate kinase and decarboxylase increased over the entire assay period. All the three enzymes showed a maximum in activity at pH 7.5. The effect of reaction temperature showed that phosphorylation increased to maximum around 35°C for mevalonate kinase and 30°C for mevalonate 5-phosphate kinase while decarboxylation rates remained constant well until 30°C temperature decreasing afterwards. The enzymes behaved differently as a function of mevalonate concentration. Mevalonate 5-phosphate formed was maximal when the initial mevalonate concentration was 272 M, whereas mevalonate 5-pyrophosphate and CO₂ were formed maximally at mevalonate concentrations of 136 M and 68M, respectively. Optimal ATP concentration in the medium was 3 mM for decarboxylase and 6 mM for kinases, and Mg⁺⁺ requirements varied from 4 mM for decarboxylase to 6 mM for kinases.     

Development and response to a diet change of some digestive enzymes in sea bass (Dicentrarchus labrax) larvae

Variations in some enzyme activities during larval development of sea bass fed live prey were investigated from hatching to day 40. Fluctuations in the enzyme specific activities (except for trypsin) occurred in three phases: initially a sharp increase until day 12, followed by a plateau and subsequently a decrease around day 23. Then activities remained constant until day 40. Trypsin activity kept rising until day 23, then fell. Enzymatic adaptation to a change in diet was studied by feeding larvae with microparticulate diet from day 25. Adaptation to dietary change was observed for amylase, alkaline phosphatase and leucine aminopeptidase, assayed in whole larvae. In larvae fed microparticulate dry diet, the activities of these three enzymes tended to be higher than in those fed natural prey. Although poor growth was observed in larvae fed microparticles, the brush border enzyme activities purified from whole body homogenate, were not impaired.