海蜇Frizzled1基因的克隆及在无性繁殖中的表达

采用RACE技术解析了海蜇(Rhopilema esculentum)Frizzled1基因的cDNA和基因组结构:Re-Fzd1基因的全长cDNA为2387 bp,其中编码区为1761bp,编码586个氨基酸的多肽。SMART分析表明,Re-Fzd1基因具备Fzd家族共同的结构特征,包括:一个由23个氨基酸组成的信号肽,一个位于N-末端富含10个保守半胱氨酸残基的半胱氨酸富集域(CRD),一个含有7个跨膜片段的跨膜结构域,以及一个含有5个重要的磷酸化位点的C端尾巴。多序列比对表明,Re-Fzd1基因与刺胞动物贝螅(Hydra echinata)、水螅(Hydra vulgaris)、半球美螅水母(Clytia hemisphaerica)和海葵(Nematostella vectensis)Fzd1具有高度相似性,与来自脊椎动物人(Homo sapiens)、鼠(Mus musculus)、爪蟾(Xenopus laevis)和斑马鱼(Danio rerio)的Fzd1、Fzd2和Fzd7家族基因也具有较高的同源性。基于N-J法,将人、鼠、爪蟾、斑马鱼和果蝇(Drosophila melanogaster)所有Fzd家族基因系统进化分析显示,除果蝇外,所有Fzd家族成员聚类成4个类群,11个亚家族,海蜇Re-Fzd1基因首先与刺胞动物门的Fzd1聚类在一起,然后与脊椎动物Fzd1、Fzd2和Fzd7三个家族聚成一个类群,表明脊椎动物Fzd1、Fzd2和Fzd7家族可能与刺胞动物门的Fzd1起源于同一个共同祖先。Re-Fzd1基因组序列中不含有内含子。实时荧光定量PCR结果显示,Re-Fzd1基因在海蜇无性繁殖的4个发育阶段均有表达,其中,表达量最高的横裂体阶段是表达量最低的稚水母阶段的3.67倍。整体原位杂交显示,在海蜇横裂体时期,Re-Fzd1原位表达在触手、基座及发生横裂的部位。这些结果都表明,Re-Fzd1不但参与了海蜇的早期发育过程,还调控了海蜇无性繁殖的发生。国家自然科学基金项目(31372507);;鳌山科技创新计划项目(2016ASKJ02);;中央级公益性科研院所基本科研业务费专项资金项目(20603022016001

Applications of stable isotope techniques in aquatic ecological studies

稳定同位素作为一种天然的示踪物,应用十分广泛,其在水域生态学中的应用也日益受到重视。生物同位素组成总是与其食物同位素组成相一致,能随食物的改变而相应地发生改变,是生物生存状况的理想指示物,为水域生态系统食物网结构与功能、物质流与能量流的研究提供了有力的技术支撑。在综评稳定同位素技术原理与方法的基础上,较为详细地对其应用于水域生态研究的理论基础与进展进行了总结。该方法的应用以水域中生产者同位素组成差异为前提,主要涉及确定食物来源、食物的贡献比例、营养级的确定、食物网结构的构建及鱼类等水生生物的洄游及迁移路线等方面,这些研究对了解生态系统的动态变化与外界环境对其影响具有重要意义。并对我国此类研究的前景和存在问题进行了探讨。Stable isotopes have been used in many research areas as natural labels and are becoming an important tool in aquatic ecological research.The theoretical basis and application of stable isotope analysis in aquatic ecology as well as its principle and method are reviewed in this paper.An organism's stable isotope ratios(δ()~(15)N and δ()~(13)C etc.) are an integration of the isotopic signatures of prey items that have been assimilated through time,the organism will come into isotopic equilibrium with its diet with the time to equilibrium depending on growth and tissue turnover rates.Stable nitrogen isotope ratios increase with trophic level by approximately 3‰ to 4‰;this provides a powerful analytical tool to quantify relative trophic position.In contrast,stable carbon isotope ratio changes very little with trophic level(0 to 1‰ enrichment per trophic level).Instead,stable carbon isotope values of organisms reflect the average δ()~(13)C of their diets.When an organism moves to a different habitat,its stable isotope ratios will change.From the stable ratios of the different habitats and the organism,we can judge the migration routine of the organism.Stable isotopes are often used to quantify the contributions of multiple sources to an organism.In general,the proportional contributions of n+1 different sources can be uniquely determined by the use of n isotope groups with linear mixing models based on mass balance equations.When there are n isotope groups and >n+1 sources,all possible combinations of each sources contribution can be obtained by a computer program(IsoSource),but the proportional contributions aren't unique.The above two cases presume that the proportional contribution of a source to an organism is the same for all isotope elements,while not all cases are so.An isotope element concentration-weighted linear mixing model was developed,whose quantified results are more reasonable,but only determined the proportional contributions of n+1 sources with n isotope groups.Prey sources,trophic level and prey proportional contributions etc.are basic information needed to construct aquatic ecosystem food web patterns and functions and material and energy flows,which are fundamental to understand the dynamic of ecosystems and effects of environment to the ecosystems.The prospect of such studies in China and some existing problems are also discussed国家自然科学基金重大资助项目(30490233)~

黄海带鱼、小带鱼RAPD和线粒体16S rRNA基因序列变异分析

对黄海带鱼、小带鱼各12个个体进行随机扩增多态DNA(RAPD)分析,对比多态位点比例、遗传多态度以及遗传距离,并构建Neighbor-joining系统树;通过PCR扩增出线粒体16SrRNA基因,纯化后直接测序,利用生物信息学方法进行序列分析和核苷酸变异比较,结合GenBank上大西洋叉尾带鱼同源序列构建UPGMA系统树.分析结果表明:(1)RAPD技术研究黄海带鱼和小带鱼的遗传多样性具有较高的灵敏度和检出率,带鱼的多态比例和遗传多态度均较小带鱼的低;(2)线粒体16S rRNA基因序列在分析这两物种遗传变异时表现出保守和变异的双重特性,种内变异极小而种间较大;(3)5个随机引物扩增出种特异的RAPD带,可作为种间分子鉴定标记;(4)研究证实带鱼和小带鱼是不同属的两个种,从而在基因水平上支持了Nelson分类系统的观点.国家重点基础研究发展规划资助项目(批准号:G19990437

基于线粒体Cytb基因的黄海、东海小黄鱼(Larimichthyspolyactis)群体遗传结构

采用线粒体dnA细胞色素b基因全序列分析技术研究了黄海、东海小黄鱼(lArIMICH-THyS POlyACTIS)的群体遗传结构.在所分析的9个取样点177个个体中,共检测到137个单倍型.9个群体呈现出高的单倍型多样性(H=0.956-1.00)和低的核苷酸多样性(π=0.0037-0.0058).单倍型邻接关系树的拓扑结构比较简单,没有明显的地理谱系结构.分子方差分析和fST显示小黄鱼的遗传变异均来自群体内个体间,而群体间无显著遗传分化.EXACT检验表明单倍型在两两群体间分布频率的差异是不显著的.中性检验和核苷酸不配对分析均表明黄海、东海的小黄鱼经历了群体扩张,扩张时间约为78—138kA前.研究结果表明,黄海、东海小黄鱼群体间具有高度的基因交流,是一个随机交配的群体.较强的扩散能力,黄海、东海的海洋环流以及近期的群体扩张可能是造成黄海、东海小黄鱼群体间遗传同质性较高的原因.国家自然科学基金(批准号:40776097);国家高技术研究发展计划(批准号:2009AA09Z401);青岛市科技计划(批准号:08-1-3-25-jch);中央级公益性科研院所基本科研业务费专项(批准号:2007-gy-06)资助项

DNA Barcoding for Cephalopoda in classification and phylogeny

应用dnA条形码通用引物扩增了11种中国近海习见头足类(CEPHAlOPOdA)共计97个个体的线粒体细胞色素C氧化酶亚基I(CyTOCHrOME COXIdASEI,COI)基因片段,与gEnbAnk收录的19种95条头足类同源序列进行比对。结果表明,头足类COI基因存在碱基插入缺失现象,杜氏枪乌贼(urOTEuTHIS duVAuCElI)插入缺失位点数多达33个;碱基组成偏倚明显,A+T含量(66.70%)显著高于g+C(33.30%)含量。基于kIMurA双参数模型计算,29个物种的种内平均遗传距离为0.0072,种间平均遗传距离(0.2024)是种内遗传距离的28.11倍。针对剑尖枪乌贼(lOlIgO EdulIS,urOTEuTHIS EdulIS,PHOTOlOlIgO EdulIS)分类和命名的分歧,dnA条形码分类结果显示,该物种与枪乌贼属(lOlIgO)和尾枪乌贼属(urOTEuTHIS)的COI基因同源性较低,不支持将其划归到lOlIgO或urOTEuTHIS。近爱尔斗蛸属(PArElEdOnE)6个代表物种的种间遗传距离较小(0.0120~0.0385),对于此类变异程度较低的物种,dnA条形码仍可准确区分,但其种间遗传距离的阈值尚待深入探讨。系统发育树的聚类分析结果表明,COI基因在种、属水平的分类鉴定及其系统进化关系与传统方法所得结果一致性较高,分别为100%、91.67%;科、目水平的一致性略低,分别为80%和66.67%。可见,线粒体COI基因作为头足类dnA条形码在物种鉴定中适用性较高,亦适用于种属水平的系统进化分析,是形态学分类系统的必要补充和佐证。The mitochondrial cytochrome c oxidase subunit Ⅰ genes (CO I) were sequenced from 97 individuals out of 11 common species of Cephalopoda in China by using the universal barcoding primers.The 97 sequences were compared with the other 95 homologous sequences belonging to 19 species of Cephalopoda obtained from the GenBank.As a result,23.33% of the Cephalopoda species' COI sequences included Insert-Deletion sites.The indels of COI sequences in Uroteuthis duvauceli counted up to 33.The COI sequences of Cephalopoda present the base preference.On average,the content of A+T (66.70%) was significantly higher than that of G+C (33.30%).As calculated by Kimera-2-parameter model,the mean distance pairwise-species and within-species was 0.202 4 and 0.007 2,respectively.The valid name and taxonomy of swordtip squid (Loligo edulis or Uroteuthis edulis or Photololigo edulis) remain controversial or unresolved.Here data gathered using COI genes does not support the assertion that swordtip squid should position in the genera of Loligo and Uroteuthis.In view of the fact that small genetic distance (0.012 0-0.038 5) among 6 species of Pareledone is found,suggesting that the threshold of barcodes (0.02) to identify species be worth furthering studies,particularly for those species with low genetic variation.According to maximum parsimony and neighbor joining trees for all 192 sequences of Cephalopoda,the consistency with morphological classification on both species and genus levels was much higher (100%,91.67%) than that of family or order levels (80%,66.67%).Consequently,providing essential revision and supplement to morphology taxonomy,the COI barcoding is a useful tool for both identification and phylogeny of Cephalopoda.国家自然科学基金项目(40776097;31061160187);公益性行业(农业)科研专项(200903005

Skeletal deformities in the larvae and juveniles of cultured tongue sole(Cynoglossus semilaevis)

采用硬骨-软骨双染色技术对半滑舌鳎(CynOglOSSuS SEMIlAEVIS günTHEr)早期发育阶段仔稚鱼全骨骼进行染色,研究其骨骼畸形发生的时间与部位及相应部位的畸形类型。实验结果表明,1--50日龄半滑舌鳎仔稚鱼的32批次495个骨骼标本中,骨骼畸形类型有14种,主要表现为脊椎骨的融合、增生、局部肥大,髓棘和脉棘的分叉和分离,尾鳍的分叉等。骨骼畸形主要发生于变态后期(25日龄)和变态后(40日龄),2个发育阶段的畸形率分别31.58%和25%。骨骼畸形的部位多见于脊柱的PH、HE和CA区,CE区尚未发现畸形。骨骼畸形会严重影响鱼类的外部形态、减缓生长及降低其市场价值。本研究初步探讨了养殖条件下半滑舌鳎骨骼畸形的发生过程,为阐明其早期发育机制及优化苗种培育和养殖条件等提供基础资料。The tongue sole(Cynoglossus semilaevis Günther) is an increasingly important marine flatfish with sig-nificant potential for aquaculture China.We documented skeletal development and deformities in tank-reared C.semilaevis larvae and juveniles(n=459).The specimens were double stained with alcian blue and alizarin red.We identified 14 types of skeletal malformations,including vertebral body fusion,hyperplasia,local hypertrophy,de-tached haemal and neural spines,bifurcated haemal and neural spines,and a bifurcated caudal fin.These skeletal malformations were most common during the post-metamorphosis stage(day 25,31.8% of specimens) and later during metamorphosis(day 40,25% of specimens).Malformations were most common in the PH(prehaemal re-gion),HE(haemal region),and CA(caudal region) of the vertebral column,but were absent in the CE(cephalic region).Our results provide insight into the developmental process and optimization of rearing conditions for C.semilaevis.国家自然科学基金项目(30871913);山东省泰山学者工程专项(2009.03-2014.02);山东省自主创新成果转化重大专项(2009ZHZX1A1201

Endocrine Hormone Adjustment Mechanism of Osmotic Pressure in Teleost

在400多科20 000多种海淡水硬骨鱼类中,有些鱼类只能生活在淡水或海水中,有些鱼类则能自由出入海淡水。这些鱼类能够在盐度变化很大的环境中生存主要依靠鳃等渗透调节器官对盐分的调节。鱼类的鳃上皮组织绝大部分为扁平细胞,但具有离子调节功能的细胞却是那些为数不多国家自然科学基金资助项目(30871913);山东省自主创新成果转化重大专项(2009ZHZX1A1201

Genetic diversity in small yellow croaker (Pseudosciaena polyactis) by RAPD analysis

小黄鱼 (Pseudosciaenapolyactis)是我国近海重要经济鱼类之一。本文分析了采自黄海和东海 5个海区共计4 8个个体小黄鱼的随机扩增DNA多态性 (RAPD)。从 4 0个 10bp引物中选取 2 0个用于群体遗传多样性分析 ,共检测出 14 5个位点 ,其中 132个 (91.0 3% )显多态性。用Shannon多样性指数量化的平均遗传多态度为 1.93(1.5 0～ 2 .4 4 ) ,群体内和群体间的遗传变异比例分别为 6 9%和 31% ;群体间的平均遗传相似度和遗传距离分别为 0 .9139和 0 .0 86 1。用非加权配对算数平均法 (UPGMA)聚类分析的结果表明 ,所分析的 5个群体可分为 3个地理群系 ,从分子水平上支持了过去有关学者把黄海和东海的小黄鱼划分为北、中、南 3个地理群系的观点Pseudosciaena polyactis is a commercially important fish species which is widely distributed in the Bohai Sea, the Yellow Sea and the northern part of the East China Sea. Forty eight individuals of P. polyactis from five sampling areas in the Yellow Sea and the East China Sea were analyzed by random amplified polymorphic DNA (RAPD) markers to determine the genetic variation among and within the stocks. A total of 145 loci were amplified using 20 random primers, of which 132 loci (91.03%) were polymorphic. Genetic diversity quantified by Shannon index varied from 1.50 to 2.44 with an average of 1.93. Partition of genetic variation indicated that 69% was distributed within stocks and 31% among stocks. The average genetic similarity and genetic distance were 0.9139 and 0.0861, respectively. Cluster analysis by UPGMA indicated that these five stocks might be divided into three groups. Results of RAPD analysis suggested extensive genetic diversity exists in this species and the genetic divergence among stocks is relatively high. The UPGMA dendrogram showed that there existed three geographic populations of P. polyactis in the Yellow Sea and the East China Sea, which supports previous conclusions based on morphological and ecological methods.国家重点基础研究专项资助项目 (G19990 43 7

Chromosome karyotype of sevenband grouper Epinephelus septemfasciatus (Thunberg,1793)

采用PHA体内直接注射法制备了七带石斑鱼(EPInEPHEluS SEPTEMfASCIATuS)头肾组织染色体标本并分析其核型。结果表明,七带石斑鱼二倍体染色体数为48,24对染色体均为端部着丝粒染色体,其染色体臂数(nf)为48,核型公式为:2n=48T,nf=48。未发现与性别相关的异型染色体,第24对染色体长度明显小于其他染色体。通过与其他22种石斑鱼染色体核型进行比较,发现七带石斑鱼具有石斑鱼属鱼类的原始核型特征,属于石斑鱼属鱼类的原始类群。The metaphase chromosomes of Epinephelus septemfasciatus were obtained from head kidney tissue by the method of PHA and colchicine injection and the karyotype was analyzed.The results showed that there were 48 telocentric chromosomes in the diploid and their karyotypic formula was 2n=48t,NF=48.The length of the 24th chromosome was distinctively shorter than that of others,while there was no obvious difference in length among the other 23 chromosomes.By comparison with the chromosome karyotypes of other 22 Epinephelus species,it can be concluded E.septemfasciatus has the representative characteristics of ancestral karyotypic pattern and belongs to the primitive grouper.青岛市科技发展计划项目(08-2-3-6-hz);农业部948项目(2008-Z8

Food competition of Engraulis japonicus and Thrissa kammalensis from the southern Yellow Sea in spring

根据南黄海鳀(Engraulis japonicus)和赤鼻棱鳀(Thrissa kammalensis)及其饵料的碳氮稳定同位素比值,采用IsoSource软件计算了两者饵料的质量贡献比。发现二者食物来源较为一致,其中仔稚鱼对鳀和赤鼻棱鳀的质量贡献率分别为42%~50%和53%~70%,是两者的首要能量与营养源。在获得食物贡献比的基础上,分别采用食物重叠指数法、聚类分析法和稳定同位素法3种方法,针对5种不同的摄食条件,依次对鳀与赤鼻棱鳀的食物重叠度进行计算。其中在饵料平均贡献比条件下,3种方法的计算结果相差不大,都在70%左右,计算偏差≤5%。研究结果表明,两者的食物竞争主要是针对仔稚鱼展开的,两者间存在显著的食物竞争关系。Stable isotopes have been used in many research fields as natural labels,and are becoming more and more appropriate option for aquatic ecological studies.An organism's stable isotope ratios(δ15N and δ13C,etc) are an integration of the isotopic signatures of preys that have been assimilated through time,the organism will come into isotopic equilibrium with its diets depending on growth and tissue turnover rates,the ratios can change with different food,are good labels of organism living conditions.According to the stable isotope ratio of an organism and its preys,we can judge its food composition.On the basis of the prey proportions of the Engraulis japonicus and Thrissa kammalensis determined by stable isotope technology.The proportions of all food sources showed that larvae and juvenile fish were the most important prey to E.japonicus and T.kammalensis,whose proportions were 42%-50% and 53%-70%,respectively.On the basis of food proportions,we calculated the prey overlap between the two fish species with three methods,diet overlap index,Cluster analysis and stable isotope technology,respectively.The mean result of each method was about 70%.There no significant difference among results of the three methods,with calculate error less than 5%.From the results of the research,we speculated that the larva and juvenile had the highest proportion,which was the main energy and trophic source of the two fish species.From the result of the research,we speculated the nitrogen stable isotope can used to calculate the food overlap of species.国家自然科学基金重大项目(30490233);; 国家自然科学基金面上项目(30570293);; 黄海水产科学研究所博士生启动基金(61200603