海洋青鳉鱼专用型基因芯片的设计及其在生态毒理学上的应用研究

以生物芯片技术为核心,结合生态毒理学与功能基因组学的方法,制备了海洋青鳉鱼专用型生物芯片,包含180个与细胞分裂、解毒反应、缺氧反应、氧化应激反应、凋亡、生长和发育、性别决定和性腺分化以及生殖激素分泌等功能相关的基因.我们提取了雌性海洋青鳉鱼成体在正常培养和缺氧处理12周后的肝脏和卵巢组织总RNA,利用自制的基因芯片进行了基因表达谱分析,结果显示海洋青鳉鱼在缺氧压力环境下,肝脏和卵巢组织的差异性表达基因分别检测出9个和6个,表明应用此类自制良好的芯片可有效获得海洋青鳉鱼的组织特异性基因表达图谱,也将加强对海洋缺氧状态、氧化应激等反应的分子生物学机制的理解.本研究对于识别异常环境状态下的基因表达模式和组织特异性标志物,进一步开展监测海洋生态毒理因素的研究奠定良好的基础.     

红鳍笛鲷(Lutjnaus erythropterus)酪氨酸酶相关蛋白1基因克隆及表达分析

本文利用RACE技术克隆获得红鳍笛鲷(Lutjnaus erythropterus)酪氨酸酶相关蛋白1基因全长,并利用生物信息学方法对该基因的m RNA序列特征、氨基酸序列特征、分类及系统发生进行了分析。结果表明,在红鳍笛鲷中,酪氨酸酶相关蛋白1基因含有TYRP1a和TYRP1b两个拷贝,其中TYRP1a序列全长3178bp,开放阅读框(ORF)长1566bp,5?端235bp,3?端1377bp;TYRP1b基因c DNA全长1871bp,ORF区长1656bp,5?端89bp,3?端126bp。序列分析发现,TYRP1a和TYRP1b基因与其它物种的同源基因保守性较高,尤其是酪氨酸酶基因家族典型的酶活性位点序列在脊椎动物中具有高度保守性。进化分析发现,TYRP1a和TYRP1b基因的种间同源性高于两个基因间的同源性。荧光定量PCR数据分析表明,TYRP1a和TYRP1b基因都在眼睛部位具有最高表达,TYRP1b在黑色皮肤也有较高表达。结果可为进一步阐述TYRP1基因在红鳍笛鲷身体和眼睛部位的色素合成机制提供一定的理论基础。     

密斑刺鲀(Diodon hystrix)gnrh基因的克隆及表达分析

密斑刺鲀(Diodonhystrix)不仅具有食用价值,同时也是传统的中药及保健食品,但目前关于密斑刺鲀的生殖内分泌调控研究较少,限制了密斑刺鲀的人工繁育基础理论的发展。总所周知,促性腺激素释放激素(gonadotropin-releasing hormone, GnRH)是生殖内分泌调控的关键因子之一。利用转录组分析及分子克隆技术,获得密斑刺鲀促性腺激素释放激素(gonadotropin-releasinghormone,GnRH) gnrh2和gnrh3基因的cDNA开放阅读框(Open Reading Frame, ORF)序列,并利用生物信息学的方法,获得密斑刺鲀gnrh2 cDNA开放阅读框共279 bp,可编码92个氨基酸残基;密斑刺鲀gnrh3cDNA开放阅读框共270 bp,可编码89个氨基酸残基。通过系统进化分析表明,密斑刺鲀GnRH与鲀科类的鱼具有较近的亲缘关系。通过聚合酶链式反应(Polymerase Chain Reaction, PCR)半定量的方法检测gnrh2和gnrh3基因在密斑刺鲀各组织中的分布,结果发现gnrh2和gnrh3基因在脾、鳃和垂体中的表达量较高,而在心脏中的表达量最低。此外,利用实时荧光定量的方法检测了密斑刺鲀gnrh2和gnrh3基因在性腺发育不同时期的表达模式,结果显示密斑刺鲀gnrh2基因在不同的性腺发育时期中没有显著性的变化,而gnrh3基因随着性腺发育成熟而不断升高。研究主要获得两个创新性的重要结果:首先是证实了密斑刺鲀存在两种gnrh基因(gnrh2和gnrh3),其次是提示了gnrh3基因可能是密斑刺鲀生殖调控的主要gnrh类型。研究结果可为深入研究密斑刺鲀生殖内分泌调控机理提供研究方向和基础。     

坛紫菜磷酸烯醇式丙酮酸羧化酶基因的克隆与序列分析

采用同源克隆的方法和RACE技术,从坛紫菜cDNA中获得了磷酸烯醇式丙酮酸羧化酶(PEPC)的序列,该段序列含2538个核苷酸包含一个终止密码子TAG和191 bp的3'端非编码区(UTR),可编码846个氨基酸.该段序列中无跨膜结构和信号肽序列,推导的氨基酸序列中C末端第813位氨基酸是丙氨酸(A),表明是一个C<,...     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

西施舌育苗水体污损生物及其防治

2000年6月-2001年8月与2002年5月-2003年8月，先后在福建长乐和广东汕头进行西施舌人工育苗的研究．发现西施舌育苗水体的污损生物21种，其中影响较大的有10种．加强水质和底质处理，改进育苗技术工艺，寻找污染源，以防为主．是治理育苗污损生物行之有效的方法．     

α2,8-唾液酸转移酶Ⅵ过量表达影响小鼠乳腺癌细胞基因表达的研究

研究α2,8-唾液酸转移酶VI(Sixth type of α2,8-sialyltransferase, ST8Sia VI) 对乳腺癌细胞生物学功能的影响及其作用的分子机制.采用全基因组芯片技术检测ST8Sia VI过表达前后小鼠乳腺癌细胞4T1基因表达谱的差异;利用PathwayExplorer分析ST8Sia VI过表达前后对基因网络的影响.实验结果表明:ST8Sia VI过表达引起201个基因表达有差异,其中22个基因与肿瘤细胞的黏附、生长、运动、免疫和周期有关.另外,PathwayExplorer分析结果显示,差异表达基因涉及的最显著变化的基因网络是"依赖β-arrestin 的G蛋白偶联受体(GPCR)信号通路";进一步的实验结果证明该通路下游Raf蛋白的磷酸化水平在ST8Sia VI过表达细胞显著升高.上述结果为ST8Sia VI生物学功能的深入研究提供了前提.     

缢蛏（Sinonovacula constricta）GST和HSP90基因克隆及其在氨氮胁迫下的表达特征分析

克隆获得缢蛏(Sinonovacula constricta)谷胱甘肽S-转移酶(Sc-GSTσ)和热休克蛋白90(Sc-HSP90)基因的cDNA全长，分析了它们的组织表达差异及其在氨氮胁迫下的表达特征。结果表明，Sc-GSTσ的全长cDNA为1 414 bp，含有639 bp的开放阅读框(Open Reading Frame，ORF)，编码212个氨基酸，Sc-GSTσ氨基酸序列与其他物种的GST氨基酸序列同源性为31.88%～43.40%；而Sc-HSP90的全长cDNA为2 752 bp，ORF为2 181 bp，编码726个氨基酸，其氨基酸序列与其他物种HSP90的氨基酸序列同源性为76.77%～87.05%。荧光定量PCR分析发现，Sc-GSTσ和Sc-HSP90在缢蛏各组织中均有表达，两者均在肝胰腺中表达量最高。氨氮胁迫后，Sc-GSTσ和Sc-HSP90 mRNA在肝胰腺中表达均显著上调(p<0.05)，表明氨氮胁迫引起机体的应激反应，2个基因可能参与机体解毒或防御过程。但胁迫后期表达量下降推测是机体的防御能力有限，不足以完全保护宿主免受应激诱导的细胞损伤。     

Risk assessment of TBT in the Japanese short-neck clam (Ruditapes philippinarum) of Tokyo Bay using a chemical fate model

A risk assessment of Tributyltin (TBT) in Tokyo Bay was conducted using the Margin of Exposure (MOE) method at the species level using the Japanese short-neck clam, Ruditapes philippinarum. The assessment endpoint was defined to protect R. philippinarum in Tokyo Bay from TBT (growth effects). A No Observed Effect Concentration (NOEC) for this species with respect to growth reduction induced by TBT was estimated from experimental results published in the scientific literature. Sources of TBT in this study were assumed to be commercial vessels in harbors and navigation routes. Concentrations of TBT in Tokyo Bay were estimated using a three-dimensional hydrodynamic model, an ecosystem model and a chemical fate model. MOEs for this species were estimated for the years 1990, 2000, and 2007. Estimated MOEs for R. philippinarum for 1990, 2000, and 2007 were approximately 1–3, 10, and 100, respectively, indicating a declining temporal trend in the probability of adverse growth effects.A simplified software package called RAMTB was developed by incorporating the chemical fate model and the databases of seasonal flow fields and distributions of organic substances (phytoplankton and detritus) in Tokyo Bay, simulated by the hydrodynamic and ecological model, respectively.     

Effects of tributyltin at environmental levels on monooxygenase system of digestive gland in hard clam Meretrix meretrix

The effects on ethoxyresorufin O-deethylase （EROD）, NADPH-cytochrome c reductase and NADH cytochrorne b5 reductase activities of digestive gland in Meretrix meretrix exposed to tributyltin （TBT） at environmental levels （0.1,1.0,10.0 ng/dm as stannum concentration）,in experimental condition, were evaluated. The EROD, NADH cytochrorne b5 reductase activities were significantly inhibited after exposure to 10.0 ng/dm^3 TBT for 8 and 20 d, the NADPH cytochrorne c reductase activities were significantly inhibited after exposure to 0.1,1.0 and 10.0 ng/dm TBT for 8 d and to 1.0 and 10.0 ng/dm for 20 d, as compared with the matched control, while NADH cytochrorne b5 reductases and NADPH cytochrome c reductase activities were induced after exposure to 10.0 ng/dm^3 TBT for 2 d. The EROD activity in the 10 ng/dm^3 group,and the NADH cytochrome b5 reductases activities in 1.0 and 10.0 ng/dm groups, were significantly induced when transferred to clean recovery tanks for 7 d. The three enzymic activities in the clams exposed to TBT were recovered to the level corresponding to that of the control group after transfer to clean recovery tanks for 20 d. NADPH cytochrome c reductase activity in Meretrix meretrix seems to be more sensitive to exposure of TBT than that of the EROD and NADH cytochrome b5 reductases. The results suggest that induction and inhibition by TBT to the monooxygenase system enzymic activity in Meretrix meretrix would simultaneously exist. The enzymic activities were inhibited by exposure for a long time. The results suggest that inhibition of the monooxygenase system should be an indication of the exposure to environmentally relevant concentrations of TBT for a long time.     

Acute toxicity of tributyltin to the Caprellidea (Crustacea: Amphipoda)

In order to compare the acute toxicity of tributyltin (TBT) between sexes, five species of caprellid amphipods were exposed to seven levels (0, 0.001, 0.01, 0.1, 1, 10 and 100 microg l(-1)) of TBT for 48 h at 20 degrees C. The 48-h LC50 values in male and female were 1.3-6.4 microg TBTCl l(-1) and 1.2-8.6 microg TBTCl l(-1), respectively. No sex-specific differences of the acute toxicity of TBT were observed in the caprellids. These findings suggest that the risk of survival in response to exposure to TBT between sexes is similar in the caprellids.     

雨生红球藻铁型超氧化物酶基因的克隆与序列分析

利用已报道的FeSOD保守区域,设计简并引物,通过简并PCR的方法获得了雨生红球藻(Haematococcus pluvialis)FeSOD(HpFeSOD)cDNA的部分序列,然后采用RACE方法分别克隆到5′端和3′端。拼接后得到HpFeSOD cDNA全长,该基因全长为1 138 bp,ORF为684 bp,编码227个氨基酸。把已经得到的HpFeSOD序列推导成氨基酸序列与一些已知物种的FeSOD氨基酸序列相比较,雨生红球藻FeSOD与杜氏藻、衣藻、团藻、石莼、颤藻的同源性为89%、83%、78%、70%和63%。分子系统学分析表明,雨生红球藻FeSOD与杜氏藻FeSOD聚在一起,介于真菌与高等植物之间并且真核微藻FeSOD与高等植物的同源性更高,推测其在进化上与高等植物的亲源关系更近。     

金乌贼热休克蛋白基因的挖掘及对低盐胁迫的响应

热休克蛋白(heat shock proteins:HSPs)为动植物响应外界胁迫产生的一类蛋白质,能有效改善机体对外界胁迫的适应能力。本研究基于先前获得的低盐胁迫金乌贼(Sepia esculenta)高通量转录组数据,对与低盐胁迫密切相关的热休克蛋白(热休克蛋白家族和晶体蛋白(crystallin)家族)基因进行挖掘;鉴于实时定量内参基因筛选,同时挖掘出金乌贼肌动蛋白(actin)家族系列基因;然后,对肌动蛋白、热休克蛋白和晶体蛋白家族基因及其编码的氨基酸序列进行生物信息学分析并探究低盐胁迫对金乌贼热休克蛋白和晶体蛋白家族基因表达的影响。金乌贼肌动蛋白家族基因包括actin、内收肌actin、胞质actin、actin 1、actin II和βactin 6个成员,热休克蛋白家族基因包括HSPβ、小HSP、HSP 10、HSP 16、HSP 30、HSP 60、HSP 70、HSP 75、HSP 90α、HSP 90和HSP 90β11个成员,金乌贼晶体蛋白家族基因包括S crystallin、S crystallin 6、S crystallin SL18和O crystallin 4个成员。氨基酸序列比对发现,肌动蛋白家族6个基因共享甘氨酸、精氨酸、丝氨酸、异亮氨酸、苏氨酸和天冬氨酸等13个位点,晶体蛋白家族4个基因共享苯丙氨酸、天冬酰胺、甘氨酸、天冬氨酸、脯氨酸、精氨酸、酪氨酸、丝氨酸和半胱氨酸等17个位点,而热休克蛋白家族HSPβ、小HSP、HSP 10、HSP 16和HSP 305个基因共享1个甘氨酸位点和4个保守位点,HSP 60、HSP 70、HSP 75、HSP 90α、HSP 90和HSP 90β6个基因仅共享2个保守位点。实时定量结果发现,低盐胁迫可以明显提高金乌贼HSPβ、HSP 16、HSP 30和HSP 70的基因表达,而明显降低晶体蛋白家族基因的表达,表明在低盐胁迫条件下,金乌贼热休克蛋白家族基因通过自身的差异表达来提高机体对外界胁迫的适应防御能力。     

基于转录组测序对两种氮素营养条件下多形微眼藻氮代谢途径的解析

探究微藻的氮代谢通路对了解其对不同氮源利用的分子机制具有重要意义。本研究利用Illumina高通量测序技术对两种氮素营养条件下(硝酸氮和尿素)多形微眼藻的转录组进行分析,通过基因功能注释及数字基因表达谱分析,研究了多形微眼藻细胞内氮代谢的调控机制。结果检测出15种参与氮代谢的酶,对应76个编码基因,构建了多形微眼藻的氮代谢通路图。其中10个酶编码基因在两种不同氮素营养条件下存在差异表达,最显著的是谷氨酸合成酶、谷氨酸脱氢酶和谷氨酰胺合成酶相关基因。有机氮源(尿素)实验组中,多形微眼藻细胞内的硝酸盐还原酶、亚硝酸盐还原酶等基因的差异表达明显高于无机氮源(硝酸钠)实验组,表明当环境中的氮源为尿素时,会对多形微眼藻细胞内硝酸盐的转化和利用有一定影响。本研究初步阐述了硝酸盐、尿素的吸收转运对多形微眼藻细胞内氮代谢的影响机制,可为硅藻在不同氮素营养条件下的吸收利用机制及氮代谢响应研究提供依据。     

Chronic toxicity of tributyltin on development and reproduction of the hermaphroditic snail Physa fontinalis: Influence of population density

Tributyltin (TBT) is toxic to aquatic organisms and occurs widely in sediments and surface waters of American and European rivers and lakes. This study investigated TBT effects on development and population growth rate (r) of the common, hermaphroditic European freshwater snail Physa fontinalis. Egg ropes of similar age (1-3 days old) were exposed to a control (solvent only) and nominal concentrations of 0.01, 1.0 and 10 microg TBT l(-1) in triplicate. Hatching and mortality were recorded during 0-40 days of exposure. At day 40, 18 juveniles were randomly selected from each concentration (i.e., six from each test vessel) and individually exposed to the same concentration of TBT in 50 ml beakers. A cohort of 20 juveniles was allowed to continue developing in the original test vessels, so that individual and grouped results could be compared. Mortality and reproduction were recorded at 48-h intervals throughout the study period (110 days). Abnormal embryonic development was observed at 1 and 10 microg TBT l(-1). Although 50% of eggs hatched at 10 microg TBT l(-1), all these hatchlings failed to survive. Survivorship of hatchlings was significantly reduced by TBT at 1 microgl(-1). In general, there was a delay in egg production in isolated snails when compared with the grouped snails. Survival, fecundity and population growth rate (r) were reduced in both individual and grouped P. fontinalis at 1.0 microg TBT l(-1). Only a decline in r was observed in snails exposed individually to 0.01 microg TBT l(-1).