黑鲷(Acanthopagrus schlegeli)肿瘤坏死因子α cDNA的克隆及特征分析

采用同源克隆的方法，从黑鲷中获得肿瘤坏死因子(TNFα)全长cDNA序列。该序列含1288个核苷酸，可编码253个氨基酸的TNF前体蛋白。它是一种跨膜蛋白，无糖基化位点和信号肽结构。黑鲷TNFα与其它鱼类的TNFα的相似性很高，占据进化上独立的分支；与哺乳类TNFα和TNFG源于共同的祖先。基因结构分析显示，该基因含有TNF家族pmfile和TNF家族的标签序列；在参与TNFα基因二硫键形成的两个半胱氨酸和TNFα三聚体形成的位点高度保守；空间结构模拟显示，它与哺乳类TNFα的空间结构相似，都是由两个β折叠片组成，每个折叠片包含5个反向平行的β链。表达研究结果表明，黑鲷TNFα在检测的各个组织中均为组成型表达，表现为在刺激与非刺激鱼体中，都可以检测到黑鲷TNFα的表达，但是其表达水平在不同组织中有很大差异。黑鲷TNFα在头肾、脾脏和鳃的表达量较高，而在心脏、肝脏、血液、肾脏和大脑中表达量较低。     

Are assemblages of black bream (Acanthopagrus butcheri) in different estuaries genetically distinct?

Samples of the estuarine-spawning teleostAcanthopagrus butcheri were collected from nine estuaries and a coastal lake, located in the Pilbara and South-western drainage divisions of Western Australia and distributed along a coastline covering a distance of nearly 2,000 km. The patterns of allozyme variation in these samples were used to explore the extent to which there was variation in the genetic compositions of black bream assemblages in geographically-isolated estuarine systems, and whether or not any such variation could be related to the geographical location or type of estuary. Although only three of 36 scorable loci (Gpi-1, Ldh andMdh-2) exhibited variation that could be used for analysis, there was considerable variation in allele frequencies at these loci among the different samples (mean F_ST=0.166). Much of the detected variation was attributable to differences between the samples collected from the two drainage divisions, which are located in very different climatic regions. Furthermore, the genetic compositions of samples from neighbouring estuaries were typically more similar to each other than to those of samples collected from more distantly-located systems. However, the assemblages in one west coast and two south coast estuaries, that are closed to the ocean for extensive periods of time during the year, all showed very similar genetic compositions. Nevertheless, it is crucial to recognise that, pairwise comparisons of samples collected from the different estuaries, both within and between the two drainage divisions, almost invariably showed statistically significant differences in allele frequencies at one or more loci. Thus, our results indicate that the local populations of black bream in individual estuaries are genetically distinct, which is probably a consequence of both a limited movement by individuals between estuaries and the effects of differences in regional and local environmental conditions.     

许氏平鲉(Sebastes schlegeli)微卫星标记开发及野生、养殖群体遗传多样性分析

对许氏平鲉进行简化基因组测序,设计微卫星引物200对,可稳定扩增的引物190对,占95%。利用一个荣成野生群体对24个多态性较高的微卫星标记进行了评价,每个位点的等位基因数(Na)为2—21个,观测杂合度(Ho)为0.0417—0.9167,期望杂合度(He)为0.0278—0.9722,多态信息含量(PIC)为0.1948—0.9496,结果显示有20个微卫星位点为中高度多态。利用这些引物对荣成野生群体和烟台养殖群体的遗传多样性进行了比较分析,野生和养殖群体的平均等位基因数(Na)分别为8.5000、6.9583,有效等位基因数(Ne)的均值分别为4.5484、3.6365,期望杂合度(He)均值分别为0.6421、0.5840,多态信息含量均值(PIC)分别为0.6088、0.5490,平均香农-威纳指数均值为1.4605、1.2834,但F检验发现无显著差异,发现两个群体的遗传多样性都处于高度多态水平,但养殖群体遗传多样性水平低于野生群体。本研究结果说明许氏平鲉的人工繁育中,通过使用较大数量的亲本进行繁育可有效防止选育群体的遗传多样性降低,但人工定向选育对选育群体的遗传多样性也产生了一定的影响。Bonferroni校正后在两个群体中各有4个位点偏离Hardy-Weinberg平衡。本研究开发的微卫星标记为许氏平鲉遗传图谱构建、分子标记辅助育种等提供了更多标记选择,对野生和养殖群体遗传多样性分析为下一步的遗传育种提供参考。     

基于SLAF-seq技术的黑棘鲷微卫星标记开发及其在鲷科鱼类中的通用性研究

黑棘鲷(Acanthopagrusschlegelii)是广泛分布于西北太平洋的暖温性中下层经济鱼类,也是我国沿海重要的海洋捕捞鱼类和增养殖对象。然而,目前有关黑棘鲷的微卫星标记研究报道较少,难以对其种质资源状况作出精确评估。本研究采用SLAF-seq技术测序共获得22489个二至六碱基重复的黑棘鲷微卫星序列,短重复序列(二、三碱基)占总微卫星序列的90.8%,长重复序列(四至六碱基)占有9.2%。经过157对随机合成引物的多态性筛选,开发出49个高多态性的黑棘鲷微卫星标记,其中短重复序列位点有25个,长重复序列位点有24个。每个位点的等位基因数(Na)为2—20(均值为8.3),观测杂合度(Ho)和期望杂合度(He)分别为0.097—0.938和0.122—0.922(均值分别为0.663和0.701),多态信息含量(PIC)为0.118—0.897(均值为0.655)。经Bonferroni校正后,有47个位点符合哈迪-温伯格平衡(HWE),各位点间未检测到连锁不平衡现象,仅2个位点偏离HWE。结果表明,所开发的大部分微卫星标记具有高多态性,蕴含的遗传信息含量较为丰富,能够为黑棘鲷的种群遗传资源评估提供数量充足、类型多样的有效分子标记。跨物种扩增结果显示,有43个黑棘鲷微卫星标记可在9种鲷科鱼类中成功扩增,其中28个标记在太平洋棘鲷(Acanthopagruspacificus)、黄鳍棘鲷(Acanthopagruslatus)和澳洲棘鲷(Acanthopagrusaustralis)中具有较好的通用性,2个标记在平鲷(Rhabdosargussarba)、蓝点赤鲷(Pagruscaeruleostictus)、真赤鲷(Pagrusmajor)、二长棘犁齿鲷(Evynnis cardinalis)及黄牙鲷(Dentex hypselosomus)中具有通用性。这些通用性标记可为阐明鲷科属、种间的系统进化关系和棘鲷属鱼类的群体遗传学分析提供新的标记来源和研究角度。     

基于线粒体控制区的中国东南沿海黄鳍棘鲷群体遗传多样性和遗传分化

本研究通过对线粒体控制区序列进行分析的技术手段,对中国宁德（ND）、厦门（XM）、漳浦（ZP）、南澳岛（NAD）、湛江（ZJ）、海口（HK）与北海（BH）的7个野生黄鳍棘鲷（Acanthopagrus latus）群体的遗传多样性和遗传分化进行比较分析。结果表明：中国东南沿海的野生黄鳍棘鲷群体呈现出中等以上的遗传多样性特征,48个黄鳍棘鲷样品的控制区序列长度为580 bp,单倍型数目为44个,总体的单倍型多样性指数（Hd）和核苷酸多样性指数（Pi）分别为0.996和0.017 30。7个野生黄鳍棘鲷群体间的遗传分化程度较低,各群体间的遗传距离为0.010 28~0.029 87。群体间的遗传分化系数（FST）为-0.068 41~0.545 86,北海群体和其他群体间存在一定程度的遗传分化。AMOVA分析显示群体内的遗传变异占比为83.34%,大部分遗传变异发生在组群内;但北海群体与其他6个群体分为两个组群后,组群间的遗传变异占比为42.66%,两个组群间存在较大程度的遗传变异。基于Kimrua距离法构建的系统进化树中显示北海群体聚为一个分支,另外6个群体聚为一支。基于TCS network构建的单倍型网络图中44个单倍型混杂在一起,未按照水系格局和地理距离进行分布,表明黄鳍棘鲷群体间未形成多个单系群。本研究可为中国东南沿海野生黄鳍棘鲷群体的种质资源利用和保护提供科学依据。     

Use of tetracycline hydrochloride and alizarin complexone for immersion marking black rockfish Sebastes schlegelii

We tested the utility of chemical marking techniques in the juvenile black rockfish Sebastes schlegelii. Juveniles(30–40 mm total length) were immersed in a range of tetracycline hydrochloride(TC) solutions at concentrations ranging from 300 to 500 mg/L, and alizarin complexone(ALC) solutions at concentrations ranging from 200 to 400 mg/L in filtered sea water(salinity of 30) for 24 h, respectively. Otoliths(sagittae, asteriscus), scales, fin rays(dorsal, pectoral, ventral, anal, and caudal fin rays), and fin spines(dorsal, ventral, and anal fin spines) were sampled and used to detect fl uorescent marks after a 60-day growth experiment. With the exception of 300 mg/L TC, acceptable marks were produced in the otoliths and fin spines by all concentrations of TC and ALC. In particular, we observed clearly visible marks in the sagittae, asteriscus, and fin spines under normal light at concentrations of 200–400 mg/L, 250–400 mg/L, and 250–400 mg/L ALC, respectively. Scales and fin rays had acceptable marks at much higher concentrations(≥350 mg/L TC, ≥250 mg/L ALC for scales and ≥350 mg/L TC, ≥300 mg/L ALC for fin rays). The best mark quality(i.e., acceptable marks were observed in all sampled structures after immersion marking) were obtained following immersion in TC at between 350–500 mg/L, and ALC between 300–400 mg/L. In addition, there was no significant difference in survival and growth of TC and ALC marked fish compared to their controls up to 60 days post-marking( P 0.05).     

黑鲷DMRT1基因cDNA片段的克隆和序列分析

根据其他鱼类DMRT1基因中的保守序列设计了一对简并引物 ,利用反转录 多聚酶链式反应 (RT PCR)的方法克隆了黑鲷 (Acanthopagrusschlegeli)DMRT1基因cDNA的一个片段 .该片段长 13 8bp ,推导的氨基酸序列由 45个氨基酸残基组成 .同源性分析表明 ,该cDNA片段与其他鱼类DMRT1基因序列具有较高的同源性     

黑鲷(Acanthopagrus schlegelii)肠道细菌群落对饲料中添加不同羽毛粉的响应

羽毛粉是一种潜在的饲料蛋白源。在水产动物饲料中添加适量的羽毛粉,不仅可以降低饲料成本,而且有助于禽类废弃物的再利用。在动物消化、降解、吸收羽毛粉过程中,动物肠道中的微生物可能发挥着重要作用;添加羽毛粉的饲料对鱼类肠道微生物多样性与群落结构的影响目前还不清楚。基于细菌16SrRNA基因高通量测序,作者对比研究了以添加羽毛粉(包括水解、酸解、膨化处理或生羽毛粉实验组,添加量为5%)和不添加羽毛粉的饲料(即对照组,包括鱼粉和商业饲料两组)喂养黑鲷12周后,黑鲷肠道细菌多样性与群落结构的变化情况。统计分析结果表明:添加羽毛粉的实验组中肠道细菌的丰富度、辛普森指数和香农指数显著低于对照组(P0.05);不含酸解羽毛粉组的实验组,其细菌群落结构与对照组差异显著(P0.05)。总体来看,黑鲷肠道细菌主要由厚壁菌门(57.3%)和变形菌门(36.3%)组成;实验组细菌群落中拟杆菌纲、梭菌纲、δ-变形菌纲、草酸杆菌科和青枯菌属的相对丰度显著低于对照组。不同的羽毛粉处理组之间也呈现出群落结构上的差异:与水解羽毛粉组相比,生羽毛粉组的变形菌门、γ-变形菌门、海洋螺菌目、盐单胞菌科和盐单胞菌属的相对丰度显著下降;与鱼粉组相比,生羽毛粉组中芽孢杆菌目在群落中的占比显著上升,而拟杆菌门、草酸杆菌科和青枯菌属的占比则显著下降,而且生羽毛粉组中厚壁菌门与变形菌门相对丰度的比值(7.42)明显高于鱼粉组(0.97)和水解羽毛粉组(0.74)且均接近显著(P=0.057)。与鱼粉组相比,水解羽毛粉组中紫单胞菌科和回线属的相对丰度显著下降,而盐单胞菌属的相对丰度显著上升。     

Use of calcein and alizarin red S for immersion marking of black rockfish Sebastes schlegelii juveniles

Black rockfi sh Sebastes schlegelii juveniles(30–40 mm total length) were immersed in a range of calcein(CAL) solutions at concentrations ranging from 50 to 250 mg/L and alizarin red S(ARS) solutions at concentrations ranging from 100 to 500 mg/L in fi ltered seawater(salinity 30) for 24 h. Fluorescent marks were detected in otoliths(sagittae, asteriscus), scales, fi n rays(dorsal, pectoral, ventral, anal, and caudal fi n rays), and fi n spines(dorsal, ventral, and anal fi n spines) after a 60-d growth experiment. With the exception of 50–100 mg/L CAL, acceptable marks were produced in the otoliths and fi n spines by all concentrations of CAL and ARS. In particular, marks were clearly visible under normal light in the sagittae, asteriscus, and fi n spines of fi sh immersed in 200–500 mg/L, 300–500 mg/L, and 200–500 mg/L ARS, respectively. Scales and fi n rays had acceptable marks at much higher concentrations(≥50 mg/L CAL, ≥300 mg/L ARS for scales and ≥50 mg/L CAL, ≥200 mg/L ARS for fi n rays). The mark quality was highest(i.e., acceptable marks were observed in all sampled structures after immersion marking) in fi sh immersed in 150–250 mg/L CAL or 300–500 mg/L ARS. In addition, there was no signifi cant difference in survival and growth of marked fi sh compared with controls 60 d post-marking( P 0.05).