缢蛏糖原磷酸化酶基因多态性与糖原含量的关联分析

为探究缢蛏糖原磷酸化酶基因(Sc-GPH)与糖原含量的关系,本研究克隆获得Sc-GPH cDNA全长,检测其在不同组织、不同月份的表达模式,并在Sc-GPH基因编码区筛选与糖原含量关联的SNP位点.结果表明,Sc-GPH cDNA全长为3963 bp,开放阅读框为2541 bp,编码846个氨基酸;氨基酸序列多重比对和系统发育树显示,缢蛏与欧洲大扇贝、虾夷扇贝、长牡蛎等贝类亲缘关系较近,而与哺乳类、甲壳类、昆虫类亲缘关系较远.实时荧光定量PCR结果显示,Sc-GPH在8个组织中均有表达,其中在外套膜和足中表达量最高(p<0.01),推测与其糖原存储能力有关;不同月份的缢蛏外套膜和足中,Sc-GPH在8月表达量最高,而此时糖原含量较低,说明Sc-GPH的表达可能受缢蛏生殖周期的影响.以缢蛏"甬乐1号"群体为实验材料,在Sc-GPH基因编码区筛选到4个与糖原含量相关SNP位点,其中c.930T>C位点在台州野生群体中得到进一步验证,该位点为高糖原缢蛏分子标记辅助选育提供了候选标记.     

缢蛏生长因子受体结合蛋白2基因克隆、时空表达及SNP筛查

为了探索生长因子受体结合蛋白2（GRB2）在缢蛏生长发育中的作用，本实验基于缢蛏转录组文库，利用SMART RACE技术克隆缢蛏GRB2（Sc-GRB2）基因的cDNA全长序列，分析其在不同组织和发育时期的表达差异，并在外显子中进行了SNP位点筛选。结果表明，Sc-GRB2基因cDNA全长1 223 bp，开放阅读框711 bp，编码236个氨基酸；氨基酸序列比对发现，缢蛏与文蛤、泥蚶等双壳贝类同源性较高（64%、59%），而与其他物种的同源性为45%~58%，表明GRB2基因比较保守；不同组织的荧光定量PCR （qRT-PCR）结果显示，Sc-GRB2基因在缢蛏7个组织中均有表达，其中足中的表达量极显著地高于其他6个组织（P<0.01）；在8个发育时期的表达差异分析发现，该基因在稚贝期表达量极显著地高于其他7个时期（P<0.01）。SNP位点筛选结果表明，在Sc-GRB2基因的外显子区域共发现11个SNP位点。     

香港牡蛎糖原合酶与糖原磷酸化酶基因多态性研究及糖原含量性状相关分子标记挖掘

香港牡蛎是我国南方沿海地区的主要经济贝类,糖原含量是衡量其品质高低的重要指标之一。本研究在转录组测序基础上,以三墩群体(SD,60只个体)和大风江群体(DFJ,90只个体)为研究对象,采用ELISA方法检测每个香港牡蛎糖原含量,并利用片段长度差异等位基因特异性PCR (Fragment length discrepant allele specific PCR,FLDAS-PCR)技术开展糖原合酶和糖原磷酸化酶基因多态性研究,开发单核苷酸多态性(Single nucleotide polymorphism,SNP)标记。结果共开发25个SNPs,其中4个SNPs (Ch_GS680,Ch_GS1248,Ch_GP992和Ch_GP1962)在实验群体中分型成功,SPSS关联分析显示SNP位点Ch_GS680与糖原含量显著关联(P<0.05),暗示其可作为香港牡蛎糖原含量性状的潜在分子标记,研究结果为高糖原含量香港牡蛎新品种的分子标记辅助选育奠定了基础。     

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。     

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。     

海湾扇贝组织蛋白酶L基因编码区的克隆和分析

通过cDNA末端快速扩增技术(RACE),从海湾扇贝(Argopecten irradians)中克隆得到了组织蛋白酶L基因(AiCL)的编码区全长,为1095 bp,推测编码364个氨基酸.经比对与分析发现蛋白序列中存在4个组织蛋白酶L活性位点保守氨基酸:Q164,C170,H309,N329;6个极为保守的半胱氨酸...     

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。     

马氏珠母贝(Pinctada fucata)组织蛋白酶L基因的克隆与表达分析

根据已构建的溶藻弧菌(Vibro alginolyticus)诱导的马氏珠母贝(Pinctada fucata)血淋巴cDNA差减文库得到的ESTs序列, 应用cDNA末端快速扩增(RACE)技术成功克隆了其组织蛋白酶L基因(PFCatL), 并对其进行了生物信息学分析; 应用实时荧光定量PCR (Real-time PCR)技术, 研究了PFCatL基因在溶藻弧菌刺激前后马氏珠母贝足、外套膜、鳃、闭壳肌等8个组织中的表达变化。结果表明, PFCatL基因cDNA全长2004bp, 其中5′非编码区(5′-UTR)50bp, 3′非编码区(3′-UTR)865bp, 开放阅读框(ORF)1089bp, 编码362个氨基酸, 其分子量计算值(MW)为40.52kDa, 理论等电点(IP)为5.20; 生物信息学分析表明, PFCatL含有16个氨基酸残基组成的信号肽序列以及组织蛋白酶前体抑制功能域I29; Clustalw2多重比对发现PFCatL氨基酸序列在催化三联体Cys-His-Asn、底物结合位点以及二硫键形成相关的半胱氨酸残基位点高度保守; Real-time PCR研究发现, PFCatL在马氏珠母贝各组织中均有表达, 但各组织间的表达量存在差异, 其中以肾和闭壳肌中的表达量最高; 溶藻弧菌感染4h后, 外套膜、鳃以及血淋巴中PFCatL基因的表达较感染前显著上调。     

缢蛏天然抗性相关巨噬蛋白2(Sc-Nramp2)基因克隆及其在副溶血弧菌刺激下的表达分析

克隆得到缢蛏天然抗性相关巨噬蛋白2（Sc-Nramp2）基因的cDNA全长序列3 681 bp,该基因的开放阅读框有1 776 bp,编码591个氨基酸,预测分子量为65.86 kDa;其结构具有Slc11蛋白家族的典型特征,包括有10个典型的跨膜结构和2个糖基化位点。Sc-Nramp2基因3'-UTR有2个类似于脊椎动物Nramp2中铁反应控制蛋白结合位点;同源性分析表明,Sc-Nramp2和太平洋牡蛎Nramp2-like的同源性最高为71.6%。实时荧光定量PCR结果表明,Sc-Nramp2基因在闭壳肌、外套膜、肝胰腺、斧足、水管和鳃6个组织中均有表达,其中肝胰腺中的表达量最高,其次是鳃,与其他组织均有极显著性差异（P<0.01）;注射副溶血弧菌后,肝胰腺中Sc-Nramp2基因的表达量较对照组显著上调（P<0.01）,且表达量呈现先上升后下降的趋势,在12 h时达到最大表达量,推测Sc-Nramp2基因参与了缢蛏非特异性免疫应答反应。     

长牡蛎转化生长因子受体基因多态性与生长性状和糖原含量的相关性分析

本研究采用PCR-SSCP技术,对长牡蛎(Grassostrea gigas)转化生长因子受体基因(Cg-TGF-βRⅠ)编码区单核苷酸多态性(SNP)与来自5个家系316个长牡蛎个体的生长性状(壳高、壳长、壳宽、总体量和软体部量)和糖原含量进行了关联分析。研究表明,在Cg-TGF-βRⅠ基因编码区扩增出的671bp基因片段中检测到4个SNP,其中3个SNP位点(T726C,A741G,A786G)与经济性状相关;A741G和A786G与生长性状和糖原含量均存在显著相关性(P0.05),T726C仅与壳长和软体部重有显著相关性(P0.05);3个SNP位点构建得到5个单倍型,H1(TAG)和H4(CAG)单倍型的个体在总体重和软体部重上均显著高于其它3种单倍型的个体。研究结果表明,Cg-TGF-βRⅠ基因多态性影响长牡蛎的生长性状和糖原含量,可用于以后的长牡蛎的分子标记辅助育种和遗传性状的改良。     

The effect of PCBs on glycogen reserves in the eastern oyster Crassostrea virginica.

Recent declines in Chesapeake Bay oyster populations have been attributed to disease, and reduced water quality from pollution. The stress associated with pollutant exposure may reduce energy available for growth and reproduction. Polychlorinated biphenyls (PCBs) are lipophilic contaminants that may potentially affect mobilization of lipid reserves, increasing reliance on glycogen stores, which could otherwise be utilized to supply energy for gametogenesis. Thus, PCBs may indirectly affect glycogen stores in oysters in a deleterious manner. To test for this effect, reproductively inactive oysters were exposed to PCBs by feeding individuals 0.7 g of algal paste containing 0, 0.35, or 3.5 micrograms PCBs daily for 8 weeks. Additionally, a group of oysters was exposed to PCBs (0, 0.35, and 3.5 micrograms) plus 0.3 g of non-toxic artificial sediment to examine interactive effects of sediment particles and PCBs. Adductor muscle, mantle, and gonadal tissues were analyzed for glycogen content. Results suggest that glycogen content is reduced in the adductor muscle with increasing PCB exposure, but there are no effects of PCBs in the mantle and gonadal tissues.     

The effect of PCBs on glycogen reserves in the eastern oyster Crassostrea virginica

Recent declines in Chesapeake Bay oyster populations have been attributed to disease, and reduced water quality from pollution. The stress associated with pollutant exposure may reduce energy available for growth and reproduction. Polychlorinated biphenyls (PCBs) are lipophilic contaminants that may potentially affect mobilization of lipid reserves, increasing reliance on glycogen stores, which could otherwise be utilized to supply energy for gametogenesis. Thus, PCBs may indirectly affect glycogen stores in oysters in a deleterious manner. To test for this effect, reproductively inactive oysters were exposed to PCBs by feeding individuals 0.7 g of algal paste containing 0, 0.35, or 3.5 μg PCBs daily for 8 weeks. Additionally, a group of oysters was exposed to PCBs (0, 0.35, and 3.5 μg) plus 0.3 g of non-toxic artificial sediment to examine interactive effects of sediment particles and PCBs. Adductor muscle, mantle, and gonadal tissues were analyzed for glycogen content. Results suggest that glycogen content is reduced in the adductor muscle with increasing PCB exposure, but there are no effects of PCBs in the mantle and gonadal tissues.     

海洋溴酚化合物BDDPM促进肝糖原合成及缓解胰岛素抵抗的作用研究

BDDPM是一种从海洋松节藻中分离出来的全新结构的溴酚类化合物,前期研究表明BDDPM具有良好的PTP1B蛋白酶活抑制能力。在本文中利用Hep G2细胞和C2C12细胞,研究BDDPM对肝细胞糖原合成能力的影响及对胰岛素抵抗的作用。结果表明,1μmol/L BDDPM可以在细胞水平显著激活胰岛素信号通路,提高肝糖原的合成,缓解棕榈酸诱导的胰岛素抵抗,初步具有开发成为新型抗糖尿病海洋药物的潜力。     

Survival, growth, metallothionein and glycogen levels of Nucella lapillus (L.) exposed to subchronic cadmium stress: the influence of nutritional state and prey type

Dogwhelks Nucella lapillus feed mainly on mussels and barnacles, and may experience periods of starvation. We report effects of nutritional state and prey type on the survival, growth, cadmium (Cd) accumulation, metallothionein (MT) induction and glycogen stores in N. lapillus exposed to Cd in water. Adult dogwhelks, with similar shell length (30.0+/-1.5 mm), were either starved or fed to satiation with barnacles Semibalanus balanoides, mussels Mytilus edulis or Cd-dosed M. edulis, and kept in filtered natural seawater (< 0.01 microg Cd 1(-1)) or Cd-contaminated (400 microg Cd 1(-1)) seawater for 80 days. Mortality and individual growth rate were determined. Cd, MT and glycogen were measured in different tissues. Prolonged starvation and exposure to Cd significantly reduced the survivorship of N. lapillus, but feeding could help dogwhelks to combat Cd toxicity and minimise mortality. Extended starvation also caused tissue wastage, leading to higher concentrations of Cd and MT in tissues, whereas fed animals increased in weight and had lower Cd and MT concentrations because of the tissue dilution effect. Prey type significantly affected growth rate of dogwhelks and indirectly influenced Cd accumulation, MT induction and glycogen stores. Eating mussels promoted better growth and higher glycogen reserves than eating barnacles. Individual growth rate decreased with increasing Cd accumulation. Cd-exposed survivors grew faster and consumed more than control animals, implying that these survivors may have better fitness and greater tolerance to Cd toxicity. The use of growth, condition index, MT and glycogen as biomarkers of environmental pollution are discussed. These results indicate a need to incorporate biological data including growth (or at least condition index) and prey type into biomonitoring programmes to allow sound interpretation.     

糖原含量与糖原磷酸化酶基因(GPH)和己糖激酶基因(HK)的表达在福建牡蛎生殖周期中的相关性分析

Glycogen, a polymer of glucose, is an important means of storing energy. It is degraded by glycogen phosphorylase (GPH) and hexokinase (HK), glycogen phosphorylase, and hexokinase cDNAs (Ca-GPH andCa-H...     

Model of the post-Cenozoic evolution of the cryolithozone of the shelf of the western part of the Laptev Sea

The analysis of the structure of the cryolithozone, facies, and thicknesses of the Quaternary sediments and the results of the physicochemical mathematical modeling of the modern shelf of the western part of the Laptev Sea support the influence of the Late Pleistocene glaciations on the heat conditions and the distribution of the permafrost in the area. A ～200-m thick glacier formed under aerial conditions from atmospheric precipitation represented the metamorphosed snow cover. According to the modeling, the long-living (from 60?50 to 10?4 ky) glacier reduced the thickness of the permafrost rocks in the reviewed shelf area for 280–360 m. The Holocene marine transgression additionally decreased the thickness from 50–140 m on the inner shelf to 220–350 m on the outer shelf. The modern submarine cryolithozone 450-0 m thick is wide-spread in the studied region from the coast to the shelf boundary (isobaths of 130–140 m), where it pinches out at a distance of ～380 km from the coast at a depth of ～250 m above the sea level.     

Investigation of the mechanism of the long-term evolution of the carbon cycle in the ecosystem of the Sevastopol bay

We analyze the components of the carbon system of the Sevastopol bay waters and the balance of main sediment-forming substances using the data of field investigations in 1998–2008. The interannual variations of total inorganic carbon and the equilibrium partial pressure of carbon dioxide in the bay water are noted. An increase in the flux of carbon dioxide into the bay and in the content of organic carbon in bottom sediments is revealed, and an explanation of this phenomenon is given. The priority accumulation of organic carbon in the sediments of the bay is established. We assess the interannual variation in the relative abundances of organic and inorganic carbon as an index of the carbon cycle stability.