氮磷营养盐与有机汞联合作用对微氏海链藻生长的影响

探讨在高营养盐（氮、磷、硅分别为浮游植物生长的营养盐阚值≥40、50、9倍）条件下，氮浓度及氮磷摩尔比值[n（N）／n（P）]变化、汞（甲基汞和乙基汞）对微氏海链藻（Thalassiosira weissflogii）生长的影响。结果表明，高营养盐区海链藻的生长遵循营养盐限制的唯一因子论；氮浓度和氮磷比提高促进生长，当n（N）／n（P）=64则呈抑制效应；生长初期，甲基汞和乙基汞均产生毒性抑制作用，生长中后期，适量甲基汞（浓度≤1．0μg／L）、乙基汞（浓度≤0．5μg／L）却呈兴奋效应，促进生长；汞形态不同，剂量-效应关系不同。氮和有机汞是海链藻生长的刺激因子。适度有机汞污染与氮富营养化对微氏海链藻的生长产生协同效应。     

microRNA介导雨生红球藻在高光胁迫下次级细胞壁形成和虾青素积累的分子机制

雨生红球藻(Haematococcuspluvialis)是天然虾青素的最佳来源,它可以在高光胁迫等不利条件下转变成厚壁细胞并积累大量虾青素。然而,目前从微小RNA (microRNA, miRNA)层面来揭示在高光胁迫下雨生红球藻次级细胞壁形成和虾青素合成的机理研究尚未见报道。对高光处理下三个时间点的雨生红球藻样品进行microRNA测序,共鉴定出342个已知miRNA和283个新miRNA。在这625个miRNA中,其中有206个miRNA的表达量受高光影响,这些差异表达的miRNA的靶基因参与了淀粉与蔗糖代谢、甘露糖与果糖代谢、糖酵解和萜类骨架生物合成等重要代谢通路。结合转录组结果发现,高光胁迫下有6个miRNA调控了3个和次级细胞壁形成相关靶基因的表达,有5个miRNA调控了5个和虾青素生物合成相关靶基因的表达。以上结果揭示了雨生红球藻在高光下miRNA调控次级细胞壁形成和虾青素生物合成的潜在机制,为雨生红球藻虾青素的代谢工程奠定了理论基础。     

硝基苯和间苯二酚对3 种海洋微藻的毒理效应

通过研究硝基苯和间苯二酚对海洋微藻的毒理效应,旨在提示化工废水污染对水环境破坏的严重性,揭示海洋微藻类净化废水的潜在应用。本试验用塔玛亚历山大藻(Alexandrium tamarense)、海链藻(Thalassiosira weissflogii)和绿色巴夫藻(Pavlova viridis)为供试藻种,以微藻生长量及叶绿素 a 的变化为参数,研究不同质量浓度的硝基苯和间苯二酚对3种海洋微藻的毒理效应。结果表明：硝基苯对塔玛藻和巴夫藻的生长具有一定的抑制作用,质量浓度为12mg/L时抑制率分别为22.9%和20.04%,但对海链藻的生长在一定程度上有促进作用。间苯二酚对海链藻和巴夫藻的生长具有抑制作用,质量浓度为12mg/L 时其抑制率分别为26.11%和23.6%,对塔玛亚历山大藻的生长有一定的促进作用。对塔玛亚历山大藻和巴夫藻而言,在硝基苯质量浓度为3mg/L 时,叶绿素 a 含量均达到最低,其叶绿素 a含量分别为对照组的68.6%和50.9%,对于海链藻,硝基苯对其叶绿素a的影响波动不大。间苯二酚对3种海洋微藻叶绿素 a 含量的影响为绿色巴夫藻>海链藻>塔玛亚历山大藻,其叶绿素 a 含量分别是对照组的1.1%,24.9%,88%。     

条斑紫菜酵母双杂交文库的构建及PyMAPK5互作蛋白的筛选

为进一步研究条斑紫菜促分裂原活化激酶家族PyMAPK5的下游互作蛋白,理解其生物学功能,本研究通过酵母双杂交的方法进行其相互作用蛋白的筛选。提取不同温度和失水逆境胁迫下的RNA,利用Invitrogen体系构建条斑紫菜酵母双杂交cDNA文库,其库容为1.44×107CFU,重组率为91.8%。以pGBKT7-PyMAPK5为诱饵蛋白载体,利用共转化方法,从文库中筛选得到26个与PyMAPK5互作的候选蛋白。候选蛋白集中在光系统II相关蛋白、捕光蛋白、微管蛋白、ATP酶、GTP结合蛋白及假设蛋白等。微管蛋白、捕光蛋白、光系统II蛋白一对一验证结果为阳性,表明在酵母体内存在互作。本研究为阐明条斑紫菜PyMAPK5与其互作蛋白的关系及解析PyMAPK5下游作用机制奠定了基础。     

抗生素对浒苔形态及共附生细菌多样性的影响

本文研究了3种抗生素联合处理黄海绿潮浒苔(Ulva prolifera)形态及共附生细菌多样性的影响。将浒苔放散的配子加入到含有3种广谱抗生素(青霉素G、硫酸新霉素、多黏菌素B)的培养液中进行培养, 与对照组相比, 发现浒苔幼苗生长速度缓慢, 且细胞变小, 藻体无法形成正常形态。进一步采用16S rDNA高通量测序分析抗生素处理藻体前后的共附生细菌多样性, 发现抗生素处理后浒苔共附生细菌丰富度和多样性降低, 其中Maribacter丰度降低。将藻体与形态发生化合物thallusin(Maribacter spp. 提取物)共培养, 发现thallusin可促进藻体形态恢复和生长, 推测抗生素可通过抑制Maribacter等细菌从而影响浒苔形态和生长, 这将为浒苔共附生细菌功能的深入研究奠定一定基础。     

合浦珠母贝iPS细胞相关的转录因子的克隆及分析

转录因子Sox2、Oct4、c-Myc和KLF4在iPS细胞研究中具有重要作用,将它们加入体外培养的细胞中,可以诱导体细胞去分化成为多能干细胞。作者通过RACE-PCR技术从合浦珠母贝(Pinctada fucata)的外套膜组织中克隆获得了3个转录因子——c-Myc、Sox2及KLF4的cDNA。c-Myc全长1585bp,开放阅读框的长度为1131bp,编码376个氨基酸,蛋白结构分析表明它具有保守的HLH和Myc-N结构域。Sox2全长1908bp,开放阅读框长度为990bp,编码329个氨基酸,蛋白结构分析它具有保守的HMG-box和Soxp结构域。KLF4全长2268bp,开放阅读框长624bp,编码207个氨基酸,预测该蛋白具有两个zf-H2C2_2结构域。BLAST分析它们均与太平洋牡蛎的相关蛋白具有较高同源性,说明其与太平洋牡蛎亲缘关系更近。RT-PCR实验发现这3个基因在合浦珠母贝外套膜、足、生殖腺、内脏团、闭壳肌和鳃6种组织中均有表达,表明它们是非常保守的转录因子。本研究对于合浦珠母贝细胞系建立和研究具有启发意义。     

Effect of regional warming on the abundance of Pseudeuphausia sinica Wang et Chen (Euphausiacea) off the Changjiang River (Yangtze River) Estuary

Distribution and abundance of Pseudeuphausia sinica off the Changjiang River Estuary (30 ° 00′ - 32 ° 00′ N, 122 ° 00′ -123 ° 30 ′E), the East China Sea were studied in relation to environmental features associated with the regional warming. P. sinica is a subtropical species. Off the Changjiang River Estuary, its abundance reached maximum in summer. To examine spatial and temporal changes of P. sinica off the Changjiang River Estuary, the authors have combined all available sampling data in 1979, 1981, and 2000-2007. This database shows that a significant increase in abundances of P. sinica was observed in spring of 2000-2007 as compared with 1979, 1981. The abundance of P. sinica increased from 0.18-0.21 ind./m 3 in 1979 and 1981 to 0.68-4.00 ind./m 3 in 2000-2007. Accordingly, the sea temperature increased obviously from spring of 1979, 1981 to the 2000s. The authors further found a positive relationship between average surface temperature and average abundance of P. sinica. Regional warming, together with the release of predator induced stress due to a sharp decline in the abundance of its predator (e.g., fishes), were thought to be responsible for the increase in abundance of P. sinica in water off the Changjiang River Estuary.     

Birds, seals and the suspension culture of mussels in Bantry Bay, a non-seaduck area in Southwest Ireland

Concerns about the environmental impacts of mariculture have grown in recent years in response to the rapid expansion of the industry. The blue mussel (Mytilus edulis) is the main product of shellfish mariculture in the Northeast Atlantic and Baltic Sea, with approximately one third of the harvest cultured using suspended longlines within sheltered marine areas. The main aim of this study was to examine the interactions, and assess the impacts (if any) of mussel suspension culture on the seabird and seal community, employing a simultaneous study of culture and control sites. The study spanned a 20-month period (from November 2001 to August 2003) and encompassed six sites in Bantry Bay (Southwest Ireland).There was no significant difference in species richness between mussel and control sites. Similarly, species diversity did not significantly differ between the mussel and control sites although control sites were generally more diverse than mussel sites, the latter particularly dominated by large numbers of Laridae.Significantly higher numbers of Phalacrocoracidae, Laridae and Alcidae were recorded in mussel sites than in control sites. However, no significant difference was found between Gaviidae or common seal (Phoca vitulina) numbers in mussel and control sites. Seasonal patterns of abundance were similar in mussel and control sites, with peak numbers of most species groups occurring in spring.Mussel suspension culture does not appear to have an adverse effect on the abundance of seabirds or common seals in this area. The safe perching platforms provided by suspension culture floats, combined with a number of other factors, contribute to an increased abundance of a number of seabird species, particularly Laridae. The possible interactions between vertebrate predators and mussel suspension aquaculture are discussed and possible explanations for the increased seabird abundance observed in these areas are offered.     

溶藻弧菌体外刺激泥蚶(Tegillarca granosa)血细胞后早期转录组研究

泥蚶(Tegillarca granosa)是我国传统养殖贝类,细菌性疾病导致的死亡已成为制约泥蚶养殖业发展的重要因素,血细胞是泥蚶免疫防御的执行者,血细胞能直接吞噬异物,能通过包囊作用将异物包裹,血细胞还在炎症反应和伤口修复中发挥作用。采用RNA-seq测序技术研究了溶藻弧菌体外刺激后泥蚶血细胞3h和6h时转录组动态变化,与未受刺激血细胞相比,3h时1790个基因表达发生显著改变,包括1319个上调基因和471个下调基因;6h时3183个基因表达发生显著改变,包括2629个上调基因和554个下调基因。KEGG通路富集分析发现溶藻弧菌刺激后免疫相关多个信号通路或生物学过程发生显著改变,如吞噬体、细胞凋亡、蛋白酶体、内质网加工、局部黏附等。部分免疫相关基因表达丰度3h时变化不显著而6h时发生显著改变,部分免疫相关基因3h和6h时表达丰度均显著改变。研究结果为泥蚶血细胞早期免疫反应提供了新的见解,为泥蚶抗病机理研究提供了理论基础。     

福建漳浦凡纳滨对虾海水养殖中后期水体细菌群落多样性分析

为分析凡纳滨对虾（Litopenaeus vannamei）海水养殖过程中水体细菌群落多样性变化,本研究采用Illumina测序技术对福建漳浦县凡纳滨对虾海水养殖中后期水体样品进行16S rRNA基因高通量测序,同时进行水质参数测定。结果表明,养殖过程中,氨氮（NH₄+-N）、化学需氧量（COD）值波动较大,其他水质参数值波动较小。检测到的细菌种群归属于19个门、35个纲、80个目、135个科、254个属,说明该养殖水体中细菌群落具有高度的多样性。变形菌门和拟杆菌门是养殖期间主要优势类群,不同养殖时期细菌组成存在一定的波动,但没有明显的变化规律。蓝细菌在第67天、115天表现出高丰度,且其丰度在养殖期间变化较大（比例变化为0.7%~63.9%）。此外,益生菌和潜在致病菌丰度在养殖期间很低。冗余分析（Redundancy analysis,RDA）表明,对细菌群落影响最大的水质因子是化学需氧量与硝酸盐浓度比值（COD/NO₃-）和COD;降低溶氧可以增加细菌丰富度。总之,本研究初步揭示了对虾养殖中后期水体微生物的演替规律,发现水质因子对养殖水体微生物组成具有显著影响,研究结果为凡纳滨对虾健康养殖提供一定的参考意义。     

硅藻岩藻黄素特性与其生物合成的研究进展

岩藻黄素的化学结构包含丙二烯键、环氧基、以及共轭羰基和羟基,是一类珍稀类胡萝卜素。岩藻黄素能够和叶绿素a/c某些蛋白形成FCP复合物,存在于许多海洋藻类细胞的光合系统内,在光捕获和光传递方面起重要作用。此外,岩藻黄素在抗肥胖、抗炎、抗癌和抗氧化等方面的生理活性已经被证实。硅藻细胞是岩藻黄素的理想来源,三角褐指藻(Phaeodactylum tricornutum)是硅藻的模式藻种。本文对三角褐指藻来源岩藻黄素的研究进展进行了评述,主要包括岩藻黄素的生理特性和功能、生物合成途径以及合成中的关键酶,影响其生物合成的环境条件等,并对其未来研究进行了展望。     

条斑紫菜PyMGST3基因克隆、表达及功能分析

微粒体谷光甘肽硫转移酶(microsomal glutathione S-transferases,MGST)作为膜结合蛋白之一,具有谷光甘肽转移酶和过氧化物酶活性,在细胞及细胞器的抗氧化胁迫中扮演着重要角色,但MGST基因在紫菜(Pyropia yezoensis)中的鉴定与分析还未见报道。本文采用RACE-PCR方法首次克隆了条斑紫菜的微粒体GST基因的全长,命名为PyMGST3,同时利用生物信息学及实时定量PCR方法对该基因的序列及诱导表达特征进行了分析,并通过原核表达进一步验证了该基因的酶活性及在抗氧化胁迫中的作用。结果表明,PyMGST3基因的c DNA序列全长为681bp,其中开放阅读框长度417bp,5'-UTR长度80bp,3'-UTR长度184bp,在受到Cd~(2+)和Cu~(2+)胁迫时,上调表达;PyMGST3蛋白具有三个跨膜结构域及跨膜疏水区,与皱波角叉菜的MGST蛋白相似性最高为60%,与其它藻类的MGST蛋白的相似性较低;在大肠杆菌中表达及纯化后的PyMGST3蛋白具有谷光甘肽转移酶活性;此外,超表达PyMGST3蛋白的重组菌株提高了对抗Cd~(2+)和Cu~(2+)毒害的能力。这些结果暗示PyMGST3基因很可能在条斑紫菜遭遇重金属等引起的氧化胁迫时起到重要的保护作用。     

适用于条斑紫菜的双向电泳样品制备方法

为了建立适于条斑紫菜(Porphyra yezoensis)蛋白质组学分析的的样品提取方法, 作者以条斑紫菜叶状体为材料, 比较了饱和硫酸铵沉淀法、三氯乙酸/丙酮沉淀法和酚提取方法3 种方法对条斑紫菜叶状体总蛋白的提取效果。结果表明, 针对条斑紫菜叶状体细胞中含有大量多糖, 多酚化合物, 色素等干扰物质的特点, 通过酚提取方法能有效去除这些干扰杂质, 得到清晰、稳定、干净的双向电泳图谱和相对较多的蛋白质点数, 为提高其他大型海藻的总可溶性蛋白提取效率提供了重要参考。     

兼养小球藻在不同浓度Fe3+培养下的蛋白质组学研究

为研究小球藻(Chlorella vulgaris)对不同浓度Fe~(3+)的响应,测定了自养和乙酸兼养小球藻在不同浓度Fe~(3+)条件下的生长速率和油脂含量,比较分析了兼养小球藻在不同浓度Fe~(3+)下的蛋白质组表达差异。结果表明,兼养小球藻比自养小球藻生长速率快,积累的生物量多,在缺铁条件下中性脂含量高。蛋白质组分析显示:在缺铁条件下光合作用相关蛋白含量最低;在缺铁和高浓度铁条件下,热激蛋白、蛋白合成和糖代谢等过程相关蛋白表达都下调,表明缺铁和高浓度铁条件对小球藻的生长都是逆境条件;在缺铁条件下氨基酸代谢相关蛋白表达上调,这可能是由于NO_3~–同化下降,氨基酸合成减少,需要进行氮的回收利用。上述结果表明:在兼养条件下缺铁培养的小球藻可获得较高生物量和中性脂含量;而高浓度铁能促进小球藻生长,但不能提高中性脂含量,对藻细胞也有一定的胁迫效应。     

Similarity of electrophoretic dissolved protein spectra from coastal to pelagic seawaters

Dissolved proteins in seawater samples collected from a coastal area of Tokyo Bay, Sagami Bay and a location off the Kuroshio Current were investigated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high resolution two-dimensional electrophoresis (2-DE). Four to nine protein bands were detected in SDS-PAGE in the apparent molecular weight (MW) range from 12 kilo Dalton (kDa) to 49 kDa. The 2-DE technique distinguished 10 to 46 protein spots exhibiting isoelectric point (pI)/MW ranging 4.3–9.2/12–63 kDa. The elecrophoretic patterns were similar between the coastal and pelagic samples, as well as previously reported patterns from various pelagic areas. The close similarity of electrophoretic mobility on both SDS-PAGE and 2-DE gels indicates the compositional homogeneity of dissolved proteins in seawater throughout a broad range of marine environments. Proteinaceous dissolved organic matter (DOM) that was unresolved and smeared staining characteristics on both SDS-PAGE and 2-DE gels was first observed in Tokyo Bay waters in the present study and its possible sources are discussed. Although the two protein species, 48 kDa and 39 kDa proteins, have been identified as homologues of Porin P and low molecular weight-alkaline phosphatase of Pseudomonas aeruginosa PAO1, respectively, four strains of P. aeruginosa and two species of Pseudomonas spp. have been newly identified as the source organisms of these proteins using the N-terminal amino acid sequence data determined in previous studies.     

Effects of β-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to β-naphthoflavone (β-NF, 66 mg/kg, i.p.) elicited a 7–9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to β-NF, and generally tracked the minimal changes observed in GST–CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by β-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to β-NF.     

海洋球石藻病毒(Coccolithovirus)硫氧还蛋白(Trx)在毕赤酵母(Pichia pastoris)中的表达及其活性分析

从实验室保存的pBS-Trx重组质粒中克隆球石藻病毒EhV-Trx基因,构建毕赤酵母重组表达载体pPIC9K-EhV-Trx,将重组质粒电转化毕赤酵母GS115,诱导分泌表达并对重组蛋白进行二硫键还原酶活性分析。结果表明,EhV-Trx基因开放阅读框为591bp,编码197个氨基酸;在毕赤酵母GS115中成功诱导表达重组EhV-Trx,经SDS-PAGE分析目的蛋白分子量约为27.8kDa;重组EhV-Trx具有二硫键还原酶的活性,能有效打开胰岛素A、B两条链的二硫键,有望开发成一种新型的硫氧还蛋白脱敏制剂应用于食品安全领域。     

无乳链球菌(Streptococcus agalactiae) 荚膜多糖合成基因研究进展

无乳链球菌是一种人畜鱼共患的重要病原菌,菌体表面的荚膜多糖是公认的毒力因子,其合成过程受荚膜多糖合成基因的调控.荚膜多糖合成基因存在于荚膜多糖操纵子中,参与无乳链球菌荚膜多糖的合成启动、寡糖和多糖的聚合以及外输并锚定于菌体表面,在新型诊断技术和减毒突变株的构建方面取得良好的应用.本文首次就cps基因的基本属性、转录调节、编码蛋白及其生物功能、对荚膜多糖合成的调控机理、在血清分型和突变株构建的应用这六个方面进行深入分析和讨论,以期为GBS cps基因的新功能研究和创新应用提供理论参考.