斜带石斑鱼囊胚期胚胎和尾芽期胚胎差异表达基因的筛选及克隆

以斜带石斑鱼囊胚期胚胎和尾芽期胚胎分别作为检验组和驱动组,构建了石斑鱼囊胚期胚胎和尾芽期胚胎的抑制性差减杂交cDNA文库。以α-tubulin作为检测指标,显示差减效率分别高达28和27。分别取囊胚期胚胎和尾芽期胚胎各192和960个PCR阳性克隆进行斑点杂交,得到15个囊胚期和131个尾芽期的斑点杂交阳性克隆。测序和数据库比对分析表明,囊胚期15个阳性克隆中有11个已知基因的cDNA片段和没有同源性的4个cDNA片段;而在尾芽期的131个阳性克隆中,有123个已知基因的cDNA片段和8个没有同源性的cD-NA片段。用半定量RT-PCR技术分析了部分基因片段在胚胎发育过程中的表达规律和和组织分布情况。这些差异表达片段的呈现为进一步揭示石斑鱼胚胎发育、早期性别决定和性腺分化的分子机制奠定了基础。     

饲料精氨酸水平对斜带石斑鱼幼鱼生长和肠道形态的影响

正精氨酸作为一种鱼类生长发育所必需的碱性氨基酸,是机体合成蛋白质的重要成分[1],对肠细胞的增殖分化和维持细胞形态结构具有重要的保护作用[2]。对陆生动物如羊[2]、仔猪[3,4]和鼠[5]等的研究表明,日粮中添加精氨酸能显著提高肠道绒毛高度和隐窝深度,从而提高肠道消化吸收能力。目前,在鱼类的相关研究中,Cheng等[6]报道在饲料中添加精氨酸或谷氨酰胺能显著提高杂交条纹鲈(Motone chrysopsxMorone,saxatilis)和美国红鱼(Sciaenops ocellatus)[7]前肠和中肠的皱襞和微绒毛高度,增大肠道吸收面积。肠道是动物吸收利用精氨酸的     

Utilization of seagrass habitats by juvenile groupers and snappers in Banten Bay,Banten Province,Indonesia

Coastal development in Banten Bay, Indonesia, decreased seagrass coverage to only 1.5% of its surface area. We investigated the importance of seagrass as habitat for juvenile groupers (Serranidae) and snappers (Lutjanidae), by performing beam trawl hauls on a weekly basis in two seagrass locations and one mudflat area, and monthly trawl hauls in three different microhabitats (dense, mixed and patchy seagrass) in one of the seagrass locations. We studied the effects of location and microhabitat, as well as temporal patterns (diel, weekly and monthly) on the probability of occurrence and abundance of the most abundant grouper (Orange-spotted grouper, Epinephelus coioides) and snapper (Russell’s snapper, Lutjanus russellii). We found that both species were almost exclusively found in seagrass locations, with a preference for microhabitats of high complexity (dense and mixed microhabitats). L. russellii had a higher probability of catch and abundance during the night, most probably because of its ability to avoid the beam trawl during daytime sampling. In addition there was an effect of week and month on the presence and abundance of both species, but patterns were unclear, probably because of high fishing pressure on juvenile groupers and snappers by push net fishermen. Groupers and snappers mainly fed on abundant shrimps, and to a lesser extent on fish. Moreover, juveniles find protection against predators in seagrass, which confirmed the critical role of quantity and quality of seagrass areas for juvenile groupers and snappers in Banten Bay.     

Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression

Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4 h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro.     

中国南方海域4种石斑鱼的遗传多样性及分子系统发生关系的分析

对中国南方海域的青石斑鱼(Epinephelus awoara)、赤点石斑鱼(E.akaara)、橙点石斑鱼(E.bleekeri)及蜂巢石斑鱼(E.merra)进行了遗传多样性和分子系统发生关系的分析。对9个微卫星DNA标记的基因型测定结果进行计算,发现这4个地理分布重叠且形态相似的石斑鱼均具有较丰富的遗传多样性,其平均等位基因数、平均多态信息含量(PIC)、平均观测杂合度(Ho)及平均期望杂合度(He)的取值范围分别为(8.22±5.02)～(18.67±9.38)、(0.56±0.21)～(0.83±0.13)、(0.62±0.03)～(0.85±0.02)和(0.60±0.07)～(0.86±0.04)。经哈迪-温伯格平衡(HWE)检验,除橙点石斑鱼外,其他3种鱼的微卫星DNA位点都基本符合平衡。利用微卫星DNA标记和线粒体细胞色素b基因对这4种鱼重建的分子系统树均显示,青石斑鱼与赤点石斑鱼的亲缘关系较近。以微卫星DNA标记对所研究石斑鱼的全部个体进行贝叶斯聚类分析的结果表明,4个鱼物种之间的分化关系明确。     

处于性腺不同发育阶段斜带石斑鱼垂体的EST表达差异分析

斜带石斑鱼是重要的海产经济鱼类,在其个体发育过程中存在先雌后雄的天然性反转现象。垂体是调节生长和生殖等生理过程的重要内分泌器官。构建了斜带石斑鱼分别处于卵巢发育起始和性反转后期的垂体SMARTcDNA的质粒文库,并通过测序分别筛选到232个和258个表达序列标签(expressed sequence tags,EST)。将所得EST与GenBank数据库中的序列进行比对,结果表明,处于卵巢发育起始和性反转后期斜带石斑鱼垂体EST中,激素所占比例均为最高,分别为40.5%和34.9%。进一步比较分析了这两个性腺发育时期斜带石斑鱼垂体EST中各种激素相对表达丰度,表明生长/催乳激素家族(GH、PRL和SL)和阿黑皮素原(POMC)表达水平下降;促性腺激素α亚基(GTHα)表达水平急剧上升,促滤泡激素β亚基(FSHβ)、促黄体激素β亚基(LHβ)表达水平上升。