海带磷酸甘露糖变位酶(PMM)基因的克隆与表达分析     

张朋艳  于雪  姚建亭  段德麟 《海洋科学》2016,40(3):32-39

磷酸甘露糖变位酶(PMM)是褐藻胶和岩藻聚糖合成过程中的关键酶之一。本研究利用c DNA末端快速克隆(RACE)技术,获得2条海带PMM基因(Sjpmm1,Sjpmm2)序列。其中,Sjpmm1的开放阅读框(ORF)长759 bp,其编码的Sj PMM1为卤酸脱卤酶(HAD)超家族成员,含252个氨基酸,分子量约为28.51 k Da;而Sjpmm2的ORF长1866 bp,其编码的Sj PMM2属于磷酸己糖变位酶超家族的成员,含621个氨基酸,分子量约为66.49 k Da。海带PMM的二级结构均以?-螺旋为主。进化分析表明,Sjpmm1来自于原始真核生物,而Sjpmm2来源于质体的第一次内共生作用。实时定量PCR分析发现,海带受到高温或低温胁迫时,Sjpmm1和Sjpmm2转录水平上升,以合成岩藻聚糖抵抗环境影响。此外,利用p MAL-c5X载体对Sj PMM1进行体外表达,得到高浓度的可溶性融合蛋白,为后续的Sj PMM功能分析提供基础。  相似文献   

Application of quantitative PCR method in detection of Lymphocystis disease virus China （LCDV-cn） in Japanese flounder （Paralichthys olivaceus）     

昝金东  孙修勤  张之文  曲凌云  张进兴 《中国海洋湖沼学报》2007,25(4):418-422

Lymphocystis disease causes serious economic losses in the fish farming industry. The causative agent of the disease is Lymphocystis disease virus China (LCDV-cn), which has a wide range of hosts. Based on competitive quantitative PCR technology, we established a method to quantify the LCDV-cn in tissue. Results demonstrate that the average amount of LCDV-cn in the peripheral blood of infected flounder with evident tumors is about 106virions/ml while the average amount in those flounder with no evident tumor but cultured with the flounder with evident tumor is about 104virions/ml. No virus was found in the negative samples of flounder.  相似文献   

Fine‐scale diet of the Australian sea lion (Neophoca cinerea) using DNA‐based analysis of faeces          下载免费PDF全文

Kristian J. Peters  Kathy Ophelkeller  Nathan J. Bott  Bruce E. Deagle  Simon N. Jarman  Simon D. Goldsworthy 《Marine Ecology》2015,36(3):347-367

We applied DNA‐based faecal analysis to determine the diet of female Australian sea lions (n = 12) from two breeding colonies in South Australia. DNA dietary components of fish and cephalopods were amplified using the polymerase chain reaction and mitochondrial DNA primers targeting the short (~100 base pair) section of the 16S gene region. Prey diversity was determined by sequencing ~50 amplicons generated from clone libraries developed for each individual. Faecal DNA was also combined and cloned from multiple individuals at each colony and fish diversity determined. Diets varied between individuals and sites. Overall, DNA analysis identified a broad diversity of prey comprising 23 fish and five cephalopod taxa, including many species not previously described as prey of the Australian sea lion. Labridae (wrasse), Monacanthidae (leatherjackets) and Mullidae (goat fish) were important fish prey taxa. Commonly identified cephalopods were Octopodidae (octopus), Loliginidae (calamary squid) and Sepiidae (cuttlefish). Comparisons of fish prey diversity determined by pooling faecal DNA from several samples provided a reasonable but incomplete resemblance (55–71%) to the total fish diversity identified across individual diets at each site. Interpretation of diet based on the recovery of prey hard‐parts identified one cephalopod beak (Octopus sp.) and one fish otolith (Parapriacanthus elongatus). The present study highlights the value of DNA‐based analyses and their capabilities to enhance information of trophic interactions.  相似文献   

Further birth records of elephant seals Mirounga leonina in New Zealand (note)     

J. A. Mills  J. P. Ryder  P. W. Shaw  R. McLay 《新西兰海洋与淡水研究杂志》2013,47(4):789-791

The second and third recorded births of southern elephant seals Mirounga leonina on the mainland of New Zealand occurred at Kaikoura in mid‐October 1976. One female pup died within 14 d of birth and the other (sex unknown) disappeared from the region with its mother on 19 November 1976.  相似文献   

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

王忠良  简纪常  鲁义善  丁燏  王蓓  陈刚  吴灶和 《海洋与湖沼》2013,44(6):1604-1611

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。  相似文献   

DNA identification of Pacific bluefin tuna (Thunnus orientalis) in the New Zealand fishery     

P. J. Smith  L. Griggs  S. Chow 《新西兰海洋与淡水研究杂志》2013,47(4):843-850

Muscle samples were collected from 69 specimens identified as Pacific bluefin tuna (Thunnus orientalis) (Temminck and Schlegel, 1844) in the New Zealand Exclusive Economic Zone (EEZ) between 1990 and 2000. Identifications before 1996 were based on body size and colour of the caudal keel; later identifications were mostly based on the shape of abdominal cavity. The tissue samples were tested with a diagnostic mitochondrial DNA marker that distinguishes southern bluefin Thunnus maccoyii (Castelnau, 1872) and Pacific bluefin tuna T. orientalis; 59 specimens were confirmed as T. orientalis and 10 as T. maccoyii. Specimens recorded as Pacific bluefin tuna by the shape of the abdominal cavity were correctly identified as T. orientalis, and this character can be used to identify large specimens landed on tuna vessels. Some specimens recorded as Pacific bluefin tuna on the basis of colour and size were T. maccoyii; and early records of T. orientalis in New Zealand waters, based on these characters, are unreliable. Unusual colour patterns were reported in some specimens of T. orientalis but not T. maccoyii. The Pacific bluefin tuna T. orientalis accounted for less than 0.3% of the bluefin tuna catch in the New Zealand EEZ during the 1990s.  相似文献   

草鱼出血病病毒VP7蛋白基因的人工合成与原核表达   总被引：2，自引：0，他引：2  

韦先超  杨泽晓  王印  裴腊梅  姚学萍  汪开毓  杨水仙 《海洋与湖沼》2013,44(3):645-650

根据GenBank已发表的GCRVVP7蛋白基因序列,设计合成2条特异性引物和19条搭桥引物,通过搭桥PCR人工合成带有3处变异碱基的GCRVVP7蛋白基因整个编码框,经过酶切、连接、PCR鉴定和测序鉴定克隆到原核表达载体pColdTF中,并转化大肠杆菌BL21(DE3),进行IPTG诱导表达和表达产物的SDS-PAGE、Western-blotting分析、纯化以及反应原性的iELISA检测。结果表明,成功人工合成了长831bp的GCRV VP7蛋白基因编码框和构建了VP7蛋白基因的重组质粒pCold TF-VP7,pColdTF-VP7转化菌经IPTG(1mmol/L)诱导3—4h即可获得特异性蛋白VP7重组蛋白的高效表达,VP7重组蛋白相对分子量大小约为73.9ku,与预期大小相符,且与鼠抗GCRV血清有良好反应原性。  相似文献   

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   总被引：3，自引：0，他引：3  

董迎辉  项翔  姚韩韩  包永波  孙长森  林志华 《海洋与湖沼》2013,44(4):937-943

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。  相似文献   

基于马氏珠母贝(Pinctada fucata)血细胞全长转录组数据补体样组分的鉴定与分析     

王菁  李桂英  林楷琪  欧静  张淑瓶  王忠良 《海洋与湖沼》2022,53(1):176-186

补体系统作为先天免疫的重要组成部分,是一种复杂的限制性蛋白水解系统,其在免疫系统中发挥着重要的防御作用。为分析马氏珠母贝补体系统的组成及作用机制,使用血细胞样品进行了全长转录组测序建库、基因比对、功能注释,共挖掘到212个潜在补体样组分相关基因。补体样组分基因经同源性比对和结构域检测分析表明,检索到的基因分别编码89个含C1q结构域蛋白、57个C型凝集素蛋白、33个纤维胶凝蛋白、11个纤维蛋白原相关蛋白、8个甘露糖结合型凝集素关联丝氨酸蛋白酶、2个含硫酯蛋白(1个C3分子,1个TEP分子)、1个补体受体、2个补体因子、9个丝氨酸蛋白酶。随机选择12个补体相关基因,使用溶藻弧菌刺激前后的血细胞样品进行实时定量PCR检测其表达水平,结果显示C1q(C1q domain containing protein)、C-lectin、MBL(mannose-binding lectin)、ficolin、MASP(mannan-binding lectin serine protease)等基因均呈现出显著差异表达,表明马氏珠母贝补体系统是一个复杂的多组分效应系统,且可能通过凝集素途径或类似于凝集素途径激活补体系统的免疫作用。研究结果为进一步验证马氏珠母贝中存在的原始补体系统提供了分子生物学证据,同时对深入了解马氏珠母贝免疫防御机制,丰富和发展海洋无脊椎动物免疫学内容也具有重要理论意义。  相似文献   

青蛤(Cyclina sinensis)TLR2基因的克隆与表达分析   总被引：2，自引：2，他引：0  

任毅鹏  高晶  潘宝平  高虹  闫春财 《海洋与湖沼》2014,45(5):1037-1043

利用转录组文库分析得到青蛤(Cyclina sinensis)的Toll样受体2(Toll-like receptor 2,TLR2)基因序列,采用荧光定量PCR法分析该基因的表达过程。结果显示,青蛤TLR2基因的cDNA开放阅读框为2082bp,编码693个氨基酸,具典型的LRRs、单次跨膜区和TIR结构域。该基因在血淋巴中的表达量最高,与肝脏、鳃、外套膜、闭壳肌和性腺有显著性差异(P0.05)。在鳗弧菌和Poly I:C刺激下,青蛤血淋巴中TLR2基因3h开始升高,48h达到最大值,与对照组和空白组有极显著性差异(P0.01);藤黄微球菌刺激下,血淋巴中CsTLR2基因3h开始升高,48h亦达到最大值,实验组与对照组有显著性差异(P0.05)。  相似文献