Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas

We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500bp and based on its deduced amino acid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semi-quantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in xenobiotic biotransformation and/or steroid metabolism.     

南极微生物对低温生活污水处理效果的初步研究

为了提高低温废水处理效果,筛选：和鉴定具有显著低温生活污水降解作用的南极微生物,并对其低温生活污水的处理效果进行了初步研究。实验结果表明,在0～5℃时。南极低温酋群对有机物的降解率显著高于中温菌的去除率,南极耐冷菌对NH3-N及TP均有一定的去除率。从南极低温茼群中筛选到5株蛋白质降解功能菌,4株脂肪降解功能菌,1株淀粉降解功能菌,2株烷烃降解功能菌和1株芳香烃降解菌,并通过16SrDNA序列对它们进行了鉴定,明确其菌属。这为寻求解决低温环境中废水处理的有效方法奠定了基础。     

大珠母贝外套膜基因PMMG1的克隆、表达及其特征分析

EF-hand结构域在真核生物细胞内钙离子吸收和运输中发挥重要作用,在贝类中可能参与贝壳和珍珠质的形成。根据已报道的贝类中具有EF-hand结构域的碱基序列设计简并引物,从大珠母贝外套膜cDNA文库中筛选得到PMMG1基因(大珠母贝外套膜基因1)。PMMG1基因全长618 bp,开放读码框编码140个氨基酸,N-端的22个氨基酸肽段为信号肽。PMMG1氨基酸序列与合浦珠母贝PFMG1的一致性为56%,预测有两个EF-hand结构域。选取编码PMMG1成熟蛋白的cDNA序列插入pET-32a质粒构建表达载体,通过IPTG诱导,Ni2+-NTA亲和层析柱纯化,成功获得预期大小的融合蛋白。凝胶电泳迁移率的变化证明PMMG1蛋白具有结合Ca2+/Mg2+的活性,组织特异性表达表明PMMG1基因在外套膜的表达量远高于其他组织。大珠母贝外套膜基因PMMG1的克隆与表达研究为进一步研究该蛋白在珍珠质矿化中的作用、探讨珍珠质形成的分子生物学机制奠定了一定基础。     

Cellular responses and disease expression in oysters (Crassostrea virginica) exposed to suspended field contaminated sediments.

Exposure of oysters to water soluble fractions derived from field-contaminated sediments (FCS) containing predominantly lower molecular weight organic aromatic compounds, has been previously demonstrated to enhance pre-existing infections caused by the protozoan parasite, Perkinsus marinus (Dermo), and the prevalence of experimentally induced infections. To further explore the role of pollution on the onset and progression of disease, effects of suspended FCS from an estuarine creek in Virginia, USA, dominated by higher molecular weight polycyclic aromatic hydrocarbons (PAHs) on cellular responses and Dermo disease expression in oysters (Crassostrea virginica) were examined. Sediments were collected from a PAH polluted estuarine creek in Virginia, USA. To test effects on cellular response, oysters from Maine were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 70.2, 105, or 140 microg PAHs, respectively) for 5, 10, 20, and 40 days. Hemocyte activities and plasma lipid, protein and lactate dehydrogenase (LDH) levels were then measured. Exposure stimulated neutral red uptake, MTT reduction, and 3H-leucine incorporation in oyster hemocytes at various exposure times, but did not affect the plasma protein, lipid and LDH levels. To test effects on Dermo expression, oysters from a Dermo enzootic area, with an initial estimated infection prevalence of 39%, were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 75.0, 113, or 150 microg PAHs, respectively) for 30 days. Exposure enhanced disease expression in oysters. However, no significant change was noted in any measured cellular or humoral parameters.     

The effects of silver nanoparticles on oyster embryos

Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release following their use in various commercial formulations and biologic applications. Filter-feeding bivalve mollusks such as oysters are valuable model species for characterizing nanoparticle bioavailability and interactions with basic cellular processes. The adults release their gametes into the environment, so their embryos and larvae are also likely targets of nanoparticles. The purpose of these studies was to characterize the toxicity of metal nanoparticles on embryonic development of oysters, Crassostrea virginica and to compare the relative sensitivity of embryos to adults. Newly-fertilized oyster embryos were exposed to silver nanoparticles (AgNP) and then the percent normal development after 48 h was assessed. Studies were conducted with adult oysters in which they were also exposed to AgNP for 48 h, and the effects on lysosomal destabilization were determined. The expression of metallothionein (MT) gene expression was also assessed in both embryos and adults. Adverse effects on embryonic development were observed at concentrations similar to those that caused both statistically and biologically significant effects on lysosomal destabilization of adults. Significant increases in MT mRNA levels were observed in both embryos and adult oysters, and MT levels were highly induced in embryos. While we do not know whether the toxicity and gene expression responses observed in this study were due to the nanoparticles themselves or the Ag ions that dissociated from the nanoparticles, these kinds of basic studies are essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as well as aquatic organisms.     

生长激素与胰岛素样生长因子-Ⅰ对马氏珠母贝壳生长形成相关基因表达的影响

研究了外源生长激素与胰岛素样生长因子-Ⅰ处理对马氏珠母贝Pinctada fucata Gould胰岛素相关多肽受体基因(irr)和5种壳基质蛋白基因(nacrein、efcbp、n19、aspein及accbp)表达水平的影响。结果表明,激素处理显著提高了irr基因的表达水平(P<0.05),这与其在软体动物中作为胰岛素样生长因子受体的作用相一致。nacrein基因的表达水平在激素处理组也有显著升高(P<0.05),表明生长激素与胰岛素样生长因子-Ⅰ两种外源激素都增强了马氏珠母贝的生长代谢水平;与对照组相比,n19、aspein与accbp3个基因的表达水平在激素处理组均下调(P<0.05),说明这3个基因的表达受到激素调节通路的抑制作用。此外,研究发现aspein与accbp两个基因的表达在各个实验样本中具有极高的相关性,说明这两个基因在激素通路中可能受到同一个上游因子调控。efcbp基因表达水平在激素处理组与对照组之间表达稳定,各样本之间无显著性的变化(P>0.05)。     

长牡蛎Wnt5b基因的鉴定及在黑色素生成中的作用

Wnt基因家族编码一类保守的分泌型糖蛋白,广泛参与细胞分化与增殖、细胞运动、凋亡、免疫调控以及色素生成等生物学过程。本研究鉴定了一个长牡蛎Wnt家族基因——CgWnt5b,比较了黑、白壳色长牡蛎中CgWnt5b转录水平的表达情况,并对其在调控黑色素生成方面的功能进行初步研究。研究结果显示,CgWnt5b编码序列全长1116 bp,编码371个氨基酸。生物信息学分析预测CgWnt5b蛋白相对分子量为42 KDa,等电点为9.28。对CgWnt5b与哺乳动物Wnt基因家族19个成员进行系统发育分析,确定了CgWnt5b为哺乳动物Wnt5a和Wnt5b的同源基因。荧光实时定量PCR检测长牡蛎外套膜、鳃、消化腺、闭壳肌、性腺、唇瓣以及血淋巴组织中CgWnt5b的mRNA分布,结果显示,CgWnt5b在外套膜、闭壳肌和唇瓣组织中的相对表达水平较高,在消化腺、鳃、血淋巴和性腺组织中相对表达量较低。黑壳长牡蛎中CgWnt5b mRNA表达量显著低于白壳长牡蛎,且siRNA敲降CgWnt5b的表达后,多个黑色素生成相关酪氨酸酶基因的表达显著上调。综上,本研究在长牡蛎中鉴定了一个Wnt家族成员CgWnt5b,可能通过抑制黑色素生成关键基因TYR的表达抑制长牡蛎中黑色素的生成。     

Expression of P-glycoprotein in southeastern oysters, Crassostrea virginica

These studies provide important fundamental information regarding the expression of P-glycoprotein (p-gp) in southeastern oysters (Crassostrea virginica). Using rhodamine transport studies, p-gp activity was detected in newly fertilized embryos. A monoclonal antibody (C219) was used to evaluate p-gp expression in oyster tissues. On the basis of laboratory studies, p-gp expression tended to be higher in gill tissues than mantle tissues, and was generally not related to salinity differences. Seasonal studies were conducted with oysters collected monthly for 1 year from Lighthouse Creek, an unpolluted site. There was a general pattern of higher p-gp expression in the warmer months and lower expression in the colder months. In contrast, total gill protein concentrations decreased during the warmer months and increased during the colder months. These studies indicate that there are seasonal patterns in p-gp expression which may represent an adaptive response to natural stressors associated with summer conditions.     

EM对SBR工艺处理生活污水的强化作用

生物强化技术对于改善现有污水处理工艺的效果具有重要作用 。实验研究以生活污水中添加有效微生物群(EM)和单以生活污水两种方式培养活性污泥,然 后分别将其引入SBR反应器,以考察EM对SBR工艺处理生活污水的强化作用。结果表明,在正 常生活污水浓度和最优工艺条件(污水pH6～8,曝气2h,静置沉淀0.5h)下,EM-SBR反应器对 污水CODcr、NH+4-N的平均去除率分别比普通SBR工艺高19.08%和23.17%;且具有较好 的稳定性。此外,EM强化的SBR工艺还具有极强的抗冲击负荷能力,当进水CODcr为2738mg．L-1时,处理3h后,出水CODcr即可达到《污水综合排放标准》的要求,去除率高于96 %,而普通SBR工艺即使处理6h也不能达标。     

中国牡蛎产业的嬗变——新认知、新品种和新产品

牡蛎是近海生态系统的重要成员,也是世界性海洋主养贝类。我国牡蛎养殖历史悠久,但高质牡蛎产品的长期匮乏已成为新形势下产业转型升级的卡点。在国际海鲜市场,高质牡蛎就意味着品质好、品相优。为实现产业高质量发展,研究了中国经济牡蛎物种组成和地理分布;揭示了温度是不同尺度种性形成的重要环境驱动因子之一;构建了首个贝类全基因组序列精细图谱,发现基因组的高变异性和基因家族的特异性扩张是种性形成的重要遗传基础;对全球27个长牡蛎群体487个个体进行全基因组深度重测序,构建了50M级单核苷酸多态(SNP)资源库并制成190k高密度SNP分型芯片。这些资源基因组学(Resourceomics)研究为高质牡蛎创制奠定了基础。其次进一步查清了牡蛎经济性状的遗传力及表型相关性,锚定糖原含量调控的基因组模块区域,建立品质性状基因模块选育技术,育成"海蛎1号"新品种,糖原含量提高25.37%,比传统育种效率提高65.81%,实现了单一营养物质的定向选育,破解了牡蛎肉质改良的世界性难题。所建立的基因模块育种技术使高质牡蛎遗传创制成为可能。在育成新品种的基础上,还利用牡蛎附着变态阶段的生物学特性及上升流和下降流的物理学...     

广东雷州半岛东部沿岸潮间带常见牡蛎的种类及其分布

在形态分类的基础上,采用多重PCR(Multiplex Species-Specific PCR)和DNA序列分析,基于细胞色素氧化酶Ⅰ(cytochromeoxidaseI,COI)基因构建系统进化树鉴定牡蛎种类及确定其分类地位,研究了广东雷州半岛东部沿岸牡蛎科(Ostreidae)常见牡蛎的种类及分布。本次研究从雷州半岛东部沿岸的11个地点采集了363个野生牡蛎样品,并对全部样品进行了形态分析和多重PCR的鉴定,对未鉴定出来的114个样品进行COI基因的扩增并测序,构建系统进化树进行种类分析,共检测出3属5种牡蛎。巨蛎属(Crassostrea)有两种,分别为熊本牡蛎(C.sikamea)185个和福建牡蛎(C.gigas angulata)109个;小蛎属(Saccostrea)有两种,棘刺牡蛎(S.echinata)48个和团聚牡蛎(S.malabonensis)19个,牡蛎属未定种Ostreasp.1有2个。结果表明,雷州半岛东部沿岸牡蛎种类丰富,至少有5种牡蛎分布于潮间带,其中优势种为熊本牡蛎和福建牡蛎,分布范围比较广。小蛎属和牡蛎属牡蛎数量较少,分布比较集中。     

饵料生物生产与自然耕种及生活污水、农业废物的处理

高畦深沟种植水稻、有机蔬菜,沟中施投化粪池污水、生活废水、腐烂瓜果蔬菜、各类粪便、田间杂草、藤蔓秸秆等培养水蚯蚓、福寿螺、小球藻、水蚤等饵料生物,构成良性循环的人工生态系统,通过水生生物和农作物的作用使有机污染物在田间得到衰减和清除。这种立体生产、综合利用集约生产模式,有利于降低饵料生物生产成本,有助于解决自然耕种收获量较少、成本较高的问题,能使有机垃圾、污水处理工艺简单、费用少,并能变废为宝生产出大量的动物蛋白和植物蛋白。     

竹醋对生活污水好氧处理的强化作用初步研究

采用在生活污水好氧处理之前投加外源物质的方法 ,研究了竹醋 (Bamboo Vinegar,BV)及其主要有机成分 (乙酸 )对污水好氧处理效果的强化作用。结果表明 :1当 VBV/ V污水 的比值达到1 / 1 0 6时 ,BV对污水 CODcr的去除有极显著的强化作用 :CODcr去除率比不加竹醋的对照组提高1 0 %。但是 ,BV对污水中 NH+ 4 - N,T- P的去除率没有影响。 2乙酸单独使用亦可显著提高污水CODcr去除率 ,当 V乙酸 / V污水 为 1 / 1 0 7时 ,强化效果最好 ,CODcr去除率提高 1 3%。由于乙酸具有与BV相似的作用特点 ,所以认为 BV对污水处理效果的强化作用来自其中的乙酸。为了将 BV应用于污水处理中 ,需要进一步研究确定 BV对活性污泥法、生物膜法处理污水的强化效果     

Comparison of the antioxidant defense system in Crassostrea rhizophorae and Crassostrea gigas exposed to domestic sewage discharges

Oysters Crassostrea rhizophorae and Crassostrea gigas were kept for fourteen days at four sites in S?o José, SC, Brazil, chosen according to a sewage discharge contamination gradient. Enzymatic activities of CAT, GST, G6PDH and GR were evaluated in gills and digestive glands. Higher CAT activity was observed in tissues of C. rhizophorae in response to sewage contamination possibly indicating peroxisome proliferation induction. C. gigas showed elevated G6PDH activity in digestive gland, and GR in gills, after domestic sewage exposure. In conclusion, C. rhizophorae and C. gigas showed different biochemical responses after in situ exposure to domestic sewage. However, C. rhizophorae showed more significant changes in CAT suggesting that this organism could be a better monitor to this kind of effluent.     

基于AFLP技术的海南三亚野生和养殖牡蛎遗传多样性研究

2008年夏季,从海南三亚分别采集26份养殖牡蛎和32份野生牡蛎样品(1份样品代表1个体),开展扩增片断长度多态性(AFLP)和数量性状分析,采用POPGENE 32软件计算多态性位点百分率、遗传多样性指数、Hardy-Weinberg遗传偏离指数、群体遗传相似度和遗传距离。基于AFLP分析获得的遗传距离和线粒体细胞色素氧化酶I亚基(CO I)基因序列,采用邻接法构建养殖和野生牡蛎的系统发育树。结果表明,选择性引物对牡蛎品种具有较好的鉴别效率;相比养殖牡蛎,野生牡蛎多样性丰富、品种间同源性较低、亲缘关系相差较远、遗传基础较宽;通过CO I基因序列分析可知,养殖牡蛎均为香港巨牡蛎Crassostrea hongkongensis,而野生牡蛎属于囊牡蛎属Saccostrea,主要为僧帽牡蛎Saccostrea cucullata。目前海南三亚牡蛎种质资源多样性相对丰富,引种还未对三亚自然环境的牡蛎资源造成明显影响。     

UASB和ABR在常温下处理生活污水的性能比较研究

沿海城市生活污水的排放,对海洋环境有着重要影响。通过试验,在常温条件下研究了升流式厌氧污泥床反应器(Upflow Anaerobic Sludge Blanket Reactor,简称UASB)与厌氧折流板反应器(Anaerobic Baffled Reactor,简称ABR)处理生活废水的性能。试验结果表明:UASB与ABR对生活污水都有良好的处理效果,在HRT较长时,ABR反应器对COD和SS的去除效果优于UASB反应器;而当HRT缩短,小于12 h,UASB反应器内上升流速增加,改善了对COD的去除效果。ABR和UASB反应器对COD的去除效果都随温度的降低而降低。     

二倍体和三倍体太平洋牡蛎(Crassostrea gigas)繁殖前后生化成分及氨基酸组成的比较研究

对太平洋蛎二倍体和三倍体在繁殖前后的几种生化成分和氨基酸组成进行了比较研究，为牡蛎的生理生化研究提供了基础数据。结果显示：太平洋牡蛎的总糖，糖原，脂肪和蛋白的含量与其繁密切相关，二倍体牡蛎在繁殖后的总糖，糖原，脂肪的含量比繁殖前分别降低了24．31％，30．37％，14．91％，三倍体分别降低了9．45％，7．17％，20．29％，而二倍体和三倍体牡蛎粗蛋白的含量在繁殖后较繁殖前分别增高了14．91％，12．285，表明了糖类和脂肪是牡蛎殖能量的主要来源，繁殖前三倍体牡蛎的糖原含量比二倍体高23．30％，繁殖后二倍体糖原含量明显降低，三倍体的糖原含量比二倍体高了73．83％，表明三倍体牡蛎由于可育性低而体内一直保持较高水平的糖原含量，从而在繁殖季节也保持了鲜美的口味。二倍体太平洋牡蛎与三倍体的氨基酸组成和含量的分析比较也表明，二倍体牡蛎的氨基酸组分在繁殖前后变化较大，而三倍体的氨基酸组分相对稳定。     

熊本牡蛎、葡萄牙牡蛎和长牡蛎组织菌群构成及功能的比较分析

为探究健康二龄熊本牡蛎、葡萄牙牡蛎和长牡蛎5种组织间细菌群落组成、多样性和功能的差异,本研究利用Illumina高通量测序技术和PICRUSt2对3种牡蛎5种组织中的细菌群落构成及其潜在功能进行了比较分析。结果显示,在3种牡蛎的5种组织中共鉴定到6 020个细菌OTUs,其中3种牡蛎整体间共有的OTUs数占42.4%。3种牡蛎5种组织中的优势菌主要隶属于γ-变形菌纲（γ-proteobacteria）、α-变形菌纲（α-proteobacteria）、柔壁菌门（Tenericutes）和厚壁菌门（Firmicutes）。弧菌属（Vibrio）在熊本牡蛎的鳃、外套膜、性腺和血淋巴中的相对丰度均显著高于长牡蛎和葡萄牙牡蛎,且分别在熊本牡蛎外套膜、长牡蛎性腺和葡萄牙牡蛎血淋巴中相对丰度最高,但均在3种牡蛎肝胰腺中相对丰度最低。3种牡蛎5种组织间的菌群多样性均有所差异,其中在熊本牡蛎鳃、葡萄牙牡蛎外套膜和长牡蛎血淋巴中菌群多样性最高,而在熊本牡蛎血淋巴、葡萄牙牡蛎性腺和长牡蛎肝胰腺中菌群多样性最低。在不区分组织的情况下,3种牡蛎整体间的菌群结构存在显著差异（r=0.661,p<0.001）。此外,在熊本牡蛎肝胰腺、葡萄牙牡蛎鳃和长牡蛎血淋巴中细菌参与的能量代谢相关功能通路的相关丰度显著高于对应牡蛎的其他组织。本文结果表明,熊本牡蛎、葡萄牙牡蛎和长牡蛎组织内的菌群构成存在差异,且由细菌介导的功能也随牡蛎种类和组织类型发生改变。     

福建沿海巨蛎属牡蛎的主要种类及其分布

本实验采用了多重种类特异性PCR（multiplex species—specific PCR）技术,研究了巨蛎属（Crassostrea）牡蛎主要种类在福建沿海的分布．从沿海11个采样地点共采集了657个野生牡蛎样本,随机抽取了327个牡蛎样本进行基因组DNA的提取和线粒体COI基因的鉴定,结果发现200个个体为葡萄牙牡蛎（Crassostrea angulata）,101个个体为近江牡蛎（Crassostrea ariakensis）,20个个体为熊本牡蛎（Crassostrea sikamea）,6个个体为香港巨牡蛎（Crassostrea hongkongensis）．此次实验中未发现长牡蛎（Crassostrea gigas）．结果表明,福建沿海有葡萄牙牡蛎、近江牡蛎、熊本牡蛎和香港巨牡蛎4种巨蛎属牡蛎分布．