Measurement of the activity of multixenobiotic resistance mechanism in the common carp Cyprinus carpio

The multixenobiotic resistance (MXR) mechanism, mediated by activity of the transmembrane P-glycoprotein, represents a basic biological defence system in aquatic organisms. Here we investigate the MXR transport activity in an aquatic vertebrate, the common carp (Cyprinus carpio). We measured the accumulation rate of a model MXR substrate, the fluorescent dye rhodamine B (RB), in gills, lateral muscles, liver and bile. Results obtained using this method showed a significant increase of RB accumulation in tissues of fish exposed for I h to 3 microM RB in the presence of the model MXR inhibitors cyclosporin A (CA, 5 microM) or verapamil (VER, 10 microM), when compared with specimens exposed without inhibitors. The highest increase in RB accumulation detected in the liver (VER 54%, CA 170%) indicates that among the tissues analysed within this study, liver is the most prominent candidate organ for the functional detection of MXR activity in C. carpio.     

Molecular cloning of superoxide dismutase (Cu/Zn-SOD) from aquatic molluscs

The potential of the first line of the active oxygen-scavenging system, partial cDNA encoding Cu/Zn superoxide dismutase (SOD) was isolated in three aquatic mollusc species: Ruditapes decussatus (marine clam), Dreissena polymorpha (continental water mussel) and Bathymodiolus azoricus (hydrothermal vent mussel). These SOD cDNA fragments were amplified by PCR with degenerate oligonucleotide primers derived from the amino acid sequence conserved in the Cu/Zn-SOD from several other organisms. A partial cDNA of CuZn-SOD was obtained for R. decussates (510 bp), D. polymorpha (510 bp) and B. azoricus (195 bp). The deduced amino acid sequence showed high similarity among the three mollusc species (57-63%) and among other species (50-65%). The residues involved in coordinating copper (His-47, 49, 64, 121) and zinc (His-64, 72, 81 and Asp-84) were well conserved among the three Cu/Zn-SOD sequences.     

基于COI序列的翡翠股贻贝Perna viridis线粒体遗传特性分析及其近缘种间的系统关系探讨

应用通用引物 COIL 1490和 COIH 2198对翡翠股贻贝Perna viridis的性腺和体细胞线粒体DNA进行PCR扩增,获得661bp长度的COI基因片段,经过比对性腺与体细胞的COI片段,发现雄性性腺与体细胞COI基因均为一个单倍型,即体内只有一种线粒体DNA类型,没有发现双单性遗传现象,雌、雄性腺的COI基因片段变异率很低（0.31%）。应用PAUP构建了NJ树、MP树以及贝叶斯法构建了贝叶斯树,对股贻贝属3种间的系统关系进行了分析,结果表明,翡翠股贻贝P. viridis与P. canaliculus 和 P. perna 之间的分化与分歧年代的估算是相吻合的。     

中国沿海9种红藻hsp70基因序列及系统进化分析

本研究自山东青岛、浙江象山和江苏南通采集共9种红藻样品, 隶属于2纲、5目、6科、8属(据NCBI), 克隆各红藻hsp70 基因, 并对所获序列进行分析。利用特异性引物P1/P3扩增, 得到的目的条带约630 bp, 分析所推导的氨基酸序列发现:所获得片段均位于HSP70的ATPase结构域附近。9种红藻hsp70 序列之间的遗传距离在0.078～0.319之间, 序列相似度在73%～92%之间, 其保守性略低于HSP70蛋白;基因对A或T结尾的密码子表现出很高的偏好性, CGC与TGG这两种密码子在这9种红藻HSP70氨基酸密码子中未出现。上述表明hsp70 及HSP70密码子偏好性可应用于红藻分子系统学研究。基于多物种HSP70构建的进化树可见, Cyanidium caldarium与Cyanidioschyzon merolae strain 10D两种原始红藻的起源早于其他红藻, 紫菜次之, 本研究中9种红藻系统发生符合NCBI的描述。在真菌、藻类和植物中, 营养方式的差异可能是造成HSP70进化树分化的基本原因, 而相同形态类型的物种中, 环境适应是抗逆能力强、遗传结构稳定的物种生物分子进化的重要因素。     

Molecular analysis of complete ssu to lsu rdna sequence in the harmful dinoflagellatealexandrium tamarense (korean isolate, HY970328M)

New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellateAlexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report inAlexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison withProrocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton,A tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities ofAlexandrium genus, particularly of the tamarensis complex.     

Teasing apart activities of different types of ABC efflux pumps in bivalve gills using the concepts of independent action and concentration addition

Fluorescent dyes and inhibitor compounds are commonly used to detect activity of multixenobiotic resistance (MXR) efflux pumps in marine invertebrates. We here address the question whether compounds acting as specific inhibitors of certain mammalian transporters can be used in dye efflux assays to distinguish different transporter activities in gill tissue from a marine mussel. We quantified effects of PSC833, a specific inhibitor of mammalian P-gp (P-glycoprotein, ABCB1), and MK571, which blocks MRP (Multidrug resistance associated protein, ABCC) type transporters, on calcein-am efflux in gill tissue of Mytilus californianus. Calcein-am acts as a substrate of both P-gp and MRP. Effects of single compounds and mixtures were determined and combined effect models predicting independent action (IA) and concentration addition (CA) of the chemicals were applied. Effect values predicted by IA showed better correspondence with the experimentally obtained data. This indicates that the inhibitor compounds target different mechanisms of calcein-am efflux and points to P-gp and MRP activities in mussel gills. Our approach could be a simple way for identifying the efflux transporter types targeted by chemosensitizers, including environmentally relevant compounds, in native tissues from marine invertebrates.     

最小弧菌热不稳定型溶血素基因克隆与序列分析

根据已发表的最小弧菌热不稳定型溶血素（heat-labile hemolysin）基因核苷酸序列，设计和合成一对特异性引物，以最小弧菌96-6-3的基因组DNA为模板，PCR扩增得到热不稳定型溶血素基因片段，克隆到质粒载体pMD-18T中进行测序和分析．其结果表明扩增的热不稳定型溶血素基因片段与已发表的最小弧菌热不稳定型溶血素的同源性高达98％，扩增片段编码由446个氨基酸组成的多肽，推测分子量为50200．软件分析表明编码的氨基酸有较高的抗原性，可作为基因工程疫苗的候选基因片段．     

Preliminary investigations on vitellogenin m-RNA induction in some bioindicator Mediterranean fish species

This paper describes the development of a RT-PCR method for assaying Vtg gene expression in different marine fish as a potentially valuable and sensitive biomarker of exposure to estrogenic chemicals. The levels of Vtg mRNA have been analyzed using primers specifically designed for the various species and the procedures have been standardized relative to actine mRNA expression levels. Different species were analyzed including organisms with a great potential as bioindicators in the Mediterranean (i.e. the red mullet Mullus barbatus, the striped mullet Mugil cephalus, the European eel Anguilla anguilla) or exposed to biomagnification of halogenated hydrocarbons and with elevated commercial value (the bluefin tuna Thunnus thynnus). The analysis of vitellogenin mRNA levels has been standardized in feral fish providing suitable indications for a future development of this approach.     

Establishment of microsatellite-based triplex PCR for parentage analysis of Chinese shrimp Fenneropenaeus chinensis

Through exploring the microsatellite primers from the random genome sequences of Chinese shrimp (Fenneropenaeus chinensis), some microsatellite primers were obtained with rich polymorphic genetic information, and a triplex PCR was established using three primers (RS1101, RS0683 and H081 primers). By adjusting the final concentration of Mg2 , dNTP and primers, and using a touch-town PCR program, the optimum amplification parameters of PCR system were obtained, which could successfully amplify the three primers in a PCR reaction. In the denatured PAGE gel, the amplified DNA fragments of three primers RS1101, RS0683 and H081 could be easily identified each other. For the triplex PCR system, the PPE (probabilities of paternity exclusion) is 0.967 9, and the DP (discrimination power) is 0.999 327. Using the triplex PCR to test ten individuals of a parentage and their parents, an individual was excluded from the parentage in all of the three microsatellite loci, which might be mixed into the parentage for some unknown reason such as factitious misplay. The triplex PCR will be of great practical value in identifying the parentages of F. chinensis.     

不同饥饿时段对黑鲷(Acanthopagrus schlegeli)ghrelin 基因表达的影响

设计了黑鲷ghrelin基因的特异性引物,提取胃组织总RNA并扩增出目的片段,将PCR产物克隆到pGEM—TEasy载体,经质粒PCR扩增、酶切和测序鉴定重组质粒,构建标准曲线等,成功建立了ghrelin基因荧光实时定量PCR检测方法,以准确分析ghrelin基因在黑鲷摄食调节中的作用。运用建立的荧光实时定量PCR方法检测了不同饥饿时段（2天、7天）黑鲷胃、肠、下丘脑组织中ghrelin mRNA的表达变化。结果表明,饥饿2天（短期饥饿）,黑鲷胃、肠组织中ghrelin mRNA表达显著上升（P〈0．05）,下丘脑ghrelin mRNA表达无显著变化（P〉0．05）;饥饿7天（长期饥饿）,黑鲷胃、肠组织ghrelin mRNA表达比饥饿2天显著下降,但仍显著高于对照组,下丘脑ghrelin mRNA表达逐渐升高（P〈0．05）;在同一饥饿时间内不同组织ghrelin mRNA表达存在显著差异（P〈0．05）。黑鲷不同组织ghrelin基因的表达随不同饥饿时段呈现出不同的变化趋势,推测ghrelin基因参与了黑鲷摄食调节的生物学讨程。     

Flounder health status in the Seine Bay. A multibiomarker study.

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.     

The cytochrome P450 1A gene (CYP1A) from European flounder (Platichthys flesus), analysis of regulatory regions and development of a dual luciferase reporter gene system.

Concensus primers designed to CYP1A-conserved regions were used to amplify a 1.3 kb probe from flounder genomic DNA via polymerase chain reaction (PCR). A 14-kb clone was isolated from a flounder genomic library constructed in lambda FIXII. Of this clone, 8 kb was sequenced, including 3 kb of upstream sequence. The predicted amino acid sequence showed closest similarity to plaice CYP1A1 (98%). Gene structure conformed to the seven exons and six introns common to previous CYP1A sequences, but intron lengths were not conserved. Concensus sequences corresponding to xenobiotic and other response elements as well as TATA, CAAT and GC boxes were identified. Upstream sequence (3.5 kb) including the first exon and intron up to the putative start codon were amplified via PCR and inserted upstream of the luciferase gene in a pGL3 reporter gene construct. The HepG2 mammalian hepatoma cell line was transiently co-transfected with the flounder CYP1A reporter gene construct and the pRL-CMV internal control construct. The maximal induction upon exposure to 100 nM 3-MC was 4.4-fold in comparison with carrier-treated cells. Use of deletion constructs resulted in loss of inducibility.     

Nursery areas of red mullet (Mullus barbatus), hake (Merluccius merluccius) and deep-water rose shrimp (Parapenaeus longirostris) in the Eastern-Central Mediterranean Sea

The spatial pattern of the nursery areas of red mullet (Mullus barbatus), hake (Merluccius merluccius) (Linnaeus, 1758) and deep-water rose shrimp (Parapenaeus longirostris) (Lucas, 1846) was studied in the South Adriatic and North Ionian Seas (Eastern-Central Mediterranean) applying geostatistical techniques and data from time series trawl surveys conducted in the area. The analysed variables were: R (number of recruits/km²) and R/Tot (fraction of recruits on the total sampled population). The structural analysis showed a spatial pattern of both variables characterized by continuity on a small scale. Predictions of nursery area localization with probability of finding recruits at different threshold values were obtained through median indicator kriging. For the red mullet the nurseries were mainly identified in the South Adriatic Sea off the Gargano peninsula and between Molfetta and Monopoli within 50 m in depth. The main concentration of hake juveniles was found to be between 100 and 200 m in depth along the Gargano peninsula and between Otranto and Santa Maria di Leuca, where a nursery of deep-water rose shrimp was also detected. An overlapping depth, between 100 and 200 m, was identified for hake and deep-water rose shrimp nurseries. Protection of these areas through limitations of fishing pressure is discussed.     

梭鱼人工养殖群体与自然群体的随机扩增多态DNA(RAPD)分析

利用随机扩增多态DNA(RAPD)技术,对黄河口海域棱鱼人工养殖群体与自然群体的遗传多样性进行了分析比较,以期由分子水平了解梭鱼的种群遗传多样性背景及人为干涉因素对梭鱼种群遗传多样性造成的影响.选用OPC组20个10碱基对(bp)的随机引物,对采自河北省黄骅市的24尾野生梭鱼和15尾人工养殖梭鱼进行了分析.选出11个扩增效果稳定的引物用于群体分析,扩增结果具有较好的可重复性.11个引物共检出112个位点,其中养殖群体中有94个表现多态,多态比例为83.93%;自然群体在96个位点上表现多态,多态比例为85.71%.经计算,养殖群体的遗传多样性指数为0.2124,自然群体的遗传多样性指数为0.2271;梭鱼两群体间的相似系数为92.82%,遗传距离为0.0718.研究结果表明,目前黄河口海域梭鱼群体的遗传多样性比较丰富,人工养殖过程对其未造成明显的影响,估计这与人工养殖过程中人为干涉因素少(如育苗历史短、亲鱼来源于自然群体、无定向选择和近亲繁殖等)有关.这一结果表明,梭鱼的人工养殖业在黄河口海域有很好的发展前景.     

斑鳢(Channa maculata)微囊藻毒素去毒相关基因sGST的克隆与序列分析

采用RT-PCR技术和RACE技术成功克隆淡水鱼类斑鳢sGST基因cDNA全序列,推测得到氨基酸序列,初步分析其结构功能域及系统进化关系。结果表明,斑鳢sGST基因cDNA序列全长为898bp,编码225个氨基酸。斑鳢sGST与真鲷、金头鲷、鲽、黑头鲦、川鲽、大口黑鲈等最新定名为ρ型的sGST氨基酸同源性较高,ρ型sGST为水生生物所特有并共同占据进化树上独立的分枝;与大鼠、小鼠、人等哺乳动物sGST现有所有类型同源性均很低,并且在进化树上距离也较远,表明本研究成功克隆的sGST基因应属于ρ型,可能在鱼类等水生生物对水栖环境的适应上有重要作用。     

二温式多重PCR检测鉴别对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)的研究与应用

建立了一种同时检测对虾白斑综合征病毒（WSSV）、传染性皮下和造血器官坏死病毒（IHHNV）的二温式多重PCR技术。根据基因库中WSSV（AF369029）和IHHNV（NC002190）基因序列，设计了两对分别与WSSV和IHHNV某段保守基因序列互补的引物，用这两对引物对同一样品中的WSSV和IHHNVDNA模板进行多重PCR扩增，结果均同时得到了两条大小与实验设计相符的593bp（WSSV）和356bp（1HHNV）特异性多重PCR扩增条带，而对其他对虾疾病病原的PCR扩增结果均为阴性；敏感性测定结果表明，该多重PCR技术最低能检测到WSSV和IHHNVDNA各100pg。用该多重PCR对400份临床病料进行检测，结果230份检出WSSV，阳性率57．5％；96份检出IHHNV，阳性率24％；56份同时检出WSSV和IHHNV，阳性率14％。18份为阴性，阴性率为4．5％。结果提示了WSSV和IHHNV广泛存在于中国南方的养殖对虾中，作者建立的二温式多重PCR可以用于这两种病毒的临床快速检测和鉴别诊断。     

中国典型水域磺胺类合成药物的环境生物地球化学特征

作为广谱抗菌的人工合成药物——磺胺类(SAs)是应用最早的一类人工合成抑菌剂之一,被广泛用于人类医疗、禽畜及水产养殖等。大量磺胺类药物的应用随代谢进入水环境中,对水生生态系统和人类健康产生重要影响并构成潜在风险,截至目前,这些影响和风险并未被探明。因此,对8种典型磺胺类合成药物在我国典型水环境中的分布特征进行了阐述,评估了它们对不同水生生物的生态毒性及生态风险,并诠释了它们在生物体内的代谢及其在生态系统中的降解途径。结果表明,不同水环境中磺胺类合成药物的浓度分布差异显著,磺胺甲恶唑和磺胺嘧啶分别在水体及沉积物中的浓度和污染程度最高;水体藻类是磺胺类合成药物最敏感的水生物种,其次是甲壳类和鱼类,磺胺甲恶唑对水生生态系统构成高风险;磺胺类合成药物进入体内后被代谢成不同的产物,与母体合成药物一同进入水环境中经历降解过程;生物降解是水生生态系统中磺胺类合成药物去除的主要途径,不同种细菌、真菌及藻类均可降解磺胺类合成药物。在以后的研究中,应当进一步加强磺胺类合成药物对水生生物的慢性毒性以及合成药物混合毒性的研究,明晰水环境磺胺类合成药物的分布-代谢-传输-效应的综合过程,探析磺胺类合成药物在水生...     

中华绒螯蟹与日本绒螯蟹线粒体COI基因片段的序列比较研究

以相应引物 PCR扩增了黄河口中华绒螯蟹线粒体细胞色素氧化酶 I亚基基因 (COI)片段 ,PCR产物经 T载体连接之后进行克隆、测序 ,得到 70 9bp的碱基序列 ,其 A,T,G,C含量分别为 34.4 1% ,2 7.93% ,2 0 .0 3%和 17.6 3%。并比较它与珠江流域中华绒螯蟹 COI序列和日本绒螯蟹 COI序列的差异 ,发现黄河口中华绒螯蟹与珠江流域中华绒螯蟹 COI序列完全相同 ,而与日本绒螯蟹差异非常明显 ,70 9或 6 5 8(不计引物 )位点中核苷酸差异数为 32 ,核苷酸差异率为 4 .5 1%或 4 .86 % (不计引物 ) ,其中 2 5个位点为转换 ,7个位点为颠换。作者倾向于支持存在中华绒螯蟹和日本绒螯蟹 ,或它们为同一种的两个地理亚种的观点     

草鱼(Ctenopharyngodon idellus)呼肠孤病毒(GCRV)VP6相互作用蛋白的筛选

为筛选与草鱼呼肠孤病毒GCRV096 VP6发生相互作用的宿主蛋白,先将VP6基因的PCR扩增产物,克隆至酵母表达载体p GBKT7以构建其诱饵质粒(p GBKT7-VP6);再将酵母菌Y2HGold(含p GBKT7-VP6)与酵母菌Y187[含草鱼肾脏细胞(CIK)c DNA文库质粒]进行人工诱导融合,然后经选择培养筛选阳性克隆。结果共筛选到4株阳性克隆,经测序、生物信息学分析,分别属于两条不同的核苷酸序列,其中之一所编码的产物与GAPDH(3-磷酸甘油醛脱氢酶)同源性较高。可见,该酶极可能在GCRV096侵染宿主的初期发挥着重要作用。