半滑舌鳎(Cynoglossus semilaevis)双链RNA依赖的蛋白激酶R(PKR)基因克隆和实时定量表达分析

采用RACE方法克隆了半滑舌鳎(Cynoglossus semilaevis)PKR基因(CsPKR), 获得了CsPKR全长cDNA序列为2907bp, 其中包含1959bp的开放阅读框, 100bp的5′非编码区和848bp的3′非编码区。保守结构域分析显示推导的CsPKR氨基酸在N端存在两个特异的dsRBD(dsRNA binding domain dsRBD)结构域, 在C端具有多个保守的激酶活性位点, 包括ATP结合位点和底物配体结合位点, 以及活性环结构A-loop。系统进化树分析显示鱼类、两栖类、鸟类及哺乳类的PKR具有共同的进化起源, CsPKR与牙鲆PKR的亲缘关系最为密切。荧光定量PCR结果显示, CsPKR基因在健康鱼多个组织中广泛表达, 在脑中的表达最高, 头肾中表达最低。经鳗弧菌和淋巴囊肿病毒分别感染后, CsPKR基因在免疫相关组织中呈现上调表达趋势, 其中感染鳗弧菌12h后在血液中表达量较对照组上升了9.28倍, 在感染淋巴囊肿病毒24h后, 在肝脏中的表达达到最大, 为对照组的9.97倍。以上结果暗示CsPKR基因在半滑舌鳎响应细菌和病毒免疫应答中起重要作用。     

泥蚶(Tegillarca granosa)生长因子受体结合蛋白2(GRB2)基因的克隆与表达分析

通过已构建的泥蚶(Tegillarca granosa)转录组文库, 利用SMART RACE技术扩增得到泥蚶Tg-GRB2基因的全长cDNA序列, 并对其生物信息学、组织表达及发育阶段表达特征进行了分析。结果表明, 泥蚶Tg-GRB2 cDNA全长1283bp, 开放阅读框为708bp, 编码236个氨基酸; 蛋白结构预测显示, Tg-GRB2蛋白包含SH3-SH2-SH3三个功能域, SH2结构域由两端的α-螺旋和中间反向平行的β-折叠片构成, SH3结构域主要由β-折叠片组成, 具备典型的结构特征; 氨基酸序列比对发现, Tg-GRB2与脊椎动物的同源性为62.1%—63.8%, 而与玻璃海鞘和日本血吸虫同源性较低。qRT-PCR检测结果显示: Tg-GRB2 mRNA在泥蚶血液、斧足、鳃、外套膜、闭壳肌、内脏团6个组织中都有表达, 而在血液中的表达量极显著地高于其它组织; 在泥蚶的不同发育阶段中, Tg-GRB2 mRNA在 2—4细胞胚胎、原肠胚、担轮幼虫、D形幼虫中均有较高的表达量, 而在D形幼虫中表达量最高, 表明Tg-GRB2基因在泥蚶早期发育中发挥重要调节作用。     

塔里木盆地英买力地区原油地球化学特征与族群划分

塔里木盆地英买力地区油气成因复杂,采用多馏分、多组分化学成分分析及单体烃碳同位素分析途径,对该区海相、陆相油进行精细分析。结果表明,英买力油田主要有两大类油组,分别是南部YM2井区的海相油(I类)和北部YM7井区的陆相油(Ⅱ类),各自具有典型的海相油与陆相油的特征。进一步分析表明,两大油组内部仍有分异。将第二油组进一步分为两亚类,以中、古生界产层为主的正常黑油和重质油为一类(Ⅱa),以古近系为主的凝析油为另一类(Ⅱb)。两亚类原油正构烷烃单体烃同位素稍有差异,芳烃组成与分布具有显著差异。较之于Ⅱa原油,Ⅱb原油富含联苯系列与氧芴系列、不同类型芳烃系列中以低分子量同系物占绝对优势,其正构烷烃单体烃碳同位素更重,与相邻的羊塔克地区原油表现出一定的相似性,表明两者之间有一定的成因联系,前者可能为混源油,由位于库车坳陷中拜城凹陷提供两种陆相烃源岩。英买力地区陆相油并非以往确认的一种成因类型,可能至少由两套性质迥异的中生界烃源岩供烃。英买力地区第一类海相油(I类)尽管生物标志物特征相近,但分析原油的单体烃碳同位素具有显著差异,同样存在油气混源的可能。英买力地区海、陆相原油特征与成因的深入剖析,对于该区精细油源确认、油气成藏机制乃至油气资源评价具有重要意义。     

大口黑鲈(Micropterus salmoides)致病性维氏气单胞菌的分离鉴定及其特性分析

大口黑鲈(Micropterus salmoides)是我国重要的淡水养殖经济鱼类品种,目前包括细菌性疾病在内的诸多因素影响了其养殖业的健康发展。维氏气单胞菌是引起淡水鱼败血症和溃疡综合征的重要病原菌。从患病大口黑鲈肝脏组织中分离到1株病原菌,通过形态学观察、生理生化特性分析、16S rRNA序列和特定毒力基因分析对其进行鉴定,并通过人工回归感染试验和药敏试验分析其致病性和耐药性。结果表明该菌为致病性维氏气单胞菌(Aeromonas veronii),含有aer、act、lip、fla、gcaT和OMPA I等毒力基因;药敏试验显示其对头孢曲松、氯霉素、氟苯尼考、强力霉素、四环素、多粘菌素、复方新诺明、左氟沙星等8种抗生素高度敏感,对新霉素、恩诺沙星、庆大霉素、链霉素、阿齐霉素、环丙沙星等6种抗生素中度敏感,对其他8种抗生素具有一定耐药性;人工回归感染实验表明该菌具有较高的致病性,对大口黑鲈的半致死浓度为1.4×10⁶ CFU/mL。维氏气单胞菌是水产养殖中的条件性致病菌,研究结果揭示了该菌株的部分生物学特性,有利于丰富维氏气单胞菌属的基础数据,同时也为鲈鱼养殖过程中的病害防控提供了一定的参考依据。     

Short- and long-term response of phytoplankton to ENSO in Prydz Bay,Antarctica: Evidences from field measurements,remote sensing data and stratigraphic biomarker records

The study provides one of the first lines of evidence showing linkages between Antarctic phytoplankton abundance and composition in response to ENSO, based on historical reconstruction of sediment biomarkers. In addition to sediment biomarkers, field measured and remote sensing data of phytoplankton abundance were also recorded from Prydz Bay, Eastern Antarctica. Com-munity structure of field measured phytoplankton showed significant El Ni？o/La Ni？a-related succession during 1990 to 2002. In general, the number of algae species decreased during El Ni？o and La Ni？a years compared to normal years. Austral summer monthly variation of remotely sensed chlorophyll-a （Chl-a）, particulate organic carbon （POC）, and sea surface temperature （SST） indicated that ENSO impacted the timing of phytoplankton blooms during 2007 to 2011. Phytoplankton blooms （indicated by Chl-a and POC） preceded the increases in SST during El Ni？o years, and lagged behind the SST increases during La Ni？a years. Stratigraphic record of marine sedimentary lipid （brassicasterol, dinosterol and alkenones） biomarkers inferred that the proportions of different algae （diatoms, dinoflagellates and haptophytes） changed significantly between El Ni？o and La Ni？a events. The relative proportion of diatoms increased, with that of dinoflagellates being decreased during El Ni？o years, while it was reversed during La Ni？a years.     

溶藻弧菌dtd基因的克隆及原核表达条件优化

根据NCBI数据库中已发表的溶藻弧菌(Vibrio alginolyticus)全基因组序列合成一对特异性引物,应用聚合酶链式反应(PCR技术)扩增溶藻弧菌HY9001菌株dtd基因,将其定向克隆到原核表达载体pET-32a构建重组表达质粒pET-DTD,并对其进行诱导温度、诱导时间、诱导IPTG浓度等条件的优化,最后探究其纯化时最佳咪唑洗脱浓度。结果表明:DTD蛋白成功表达,且其以包涵体的形式存在,在诱导温度37℃、IPTG浓度0.1 mmol/L条件下诱导5 h表达量最高,纯化最佳咪唑洗脱浓度为150 mmol/L。     

平湖油气田主断裂西部烃源评价及油源对比

平湖油气田主力含油气区位于平湖主断裂以东区域,油源主要来源于东部生烃凹陷的半封闭海湾暗色泥岩和煤层.公司于2011年11月在主断裂西部钻了一口探井XY井,目的是为探测主断裂西部含油气情况,扩大油气储量.结果钻遇油气层12m,测试获日产气14.66×104m3,日产油107.8 m3,取得了一定突破.为进一步评价西部含油气规模,钻后进行了油源对比研究,认为西部的油气主要是东部运移聚集的结果,西部源岩也有一定贡献.西部有形成一定规模油气藏的潜力.     

红藻DNA条形码分析技术研究进展

DNA条形码是指利用一段相对较短的标准DNA片段,对物种进行识别和鉴定。目前该技术在动物、植物物种鉴定领域已经得到了广泛的研究和应用。在藻类的研究中,尚未确定一条统一的标准条形码基因,现阶段都是使用2条或2条以上基因序列来完成物种鉴定。对于形态多样、种类繁多的海洋红藻,常用的DNA条形码基因有COI基因(Partial cytochrome c oxidase I gene)、UPA基因(Partial 23SrRNA gene,universal plastid amplicon)、LSU基因(Partial 28SrRNA gene)和rbcL基因(The large subunit of ribulose-1,5-bisphosphate carboxylase)等,这些基因中2个或3个基因的互补运用准确有效地提高了红藻的鉴定准确率,尤其是COI基因的种间差异大足够区分相近物种。本文在概述条形码的原理及其标准的基础上,阐述了红藻DNA条形码鉴定研究的新进展以及常用几种基因片段的优缺点,并对条形码在红藻鉴定中存在的问题进行了分析,对其应用前景进行了展望。     

Phylogenetic analysis of epibacterial communities on the surfaces of four red macroalgae

Macroalgal surfaces are prone to being attached by bacteria. Epibacterial community structures on marine macroalgae are host-specific but temporally and spatially variable. In this study, we investigated the structure of epibacterial communities on the surfaces of four red macroalgae, Gracilaria lemaneiformis, Gloiopeltis furcata, Mazzaella sp. and Porphyra yezoensis, by analyzing the sequences of 16 S r RNA gene libraries. Healthy individuals of all macroalgae species were collected in winter from a farm at Dalian, China. The results showed that the epibacterial communities were mainly dominated by α-Proteobacteria, γ-Proteobacteria and Bacteroidetes. Deinococcus-Thermus, Spirochaetes and ε-Proteobacteria were also found. The majority of cloned sequences shared the greatest similarity to those of culturable organisms. A large portion of sequences from the α-Proteobacteria homed in Roseobacter clade, i.e., genera Ahrensia, Roseovarius, Litoreibacter, Octadecabacter, Thaiassobacter and Sulfitobacter, while members of Bacteroidetes mainly belonged to family Flavobacteriaceae. The cloned sequences could be separated into 66 OTUs at 0.01 distance value, and rare common OTUs were found among libraries. At genus level, Pseudoalteromonas dominated Gr. lemaneiformis and Gl. furcata libraries, accounting for 72.2% and 47.3%, respectively. Sulfitobacter dominated P. yezoensis library, accounting for 35.4%. A previously undefined cluster within Deinococcus-Thermus dominated Mazzaella sp. library, accounting for 24.6% of the all. These results indicated that a broad range of bacteria inhabited the surfaces of these macroalgae.     

Identification of five sea cucumber species through PCR-RFLP analysis

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene (COI) was used to identifing five sea cucumber species (Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.