新疆3种雅罗鱼线粒体DNA控制区序列的差异和系统进化关系

对分布在新疆的准噶尔雅罗鱼(Leuciscus merzbacheri)、贝加尔雅罗鱼(Leuciscus leuciscus baicalensis)和高体雅罗鱼(Leuciscus idus)3个鱼种共24尾个体的线粒体DNA D-loop控制区核苷酸序列进行了测定,获得24条长度为667—669bp的同源基因序列。3种雅罗鱼之间的序列差异在6．39％—9．89％之间,贝加尔雅罗鱼与高体雅罗鱼种间序列同源性高,变异程度小;贝加尔雅罗鱼与准噶尔雅罗鱼种间序列同源性最低,变异程度最大。所采集的贝加尔雅罗鱼两个地理群体(赛里木湖和额尔齐斯河)内mtDNA的平均核苷酸碱基序列差异为1．07％和1．08％;两群体间的序列差异为1．07％,显示两个地理群体间无明显分化。DNA序列数据显示,这3种鱼类线粒体DNAD-loop序列变异丰富,24尾个体呈现独自的单倍型。同源基因序列平均含AT碱基64．1％,GC碱基35．9％,显示准噶尔雅罗鱼、贝加尔雅罗鱼、高体雅罗鱼的线粒体DNAD-loop区核苷酸组成的不均一性。分子系统树提示,贝加尔雅罗鱼与高体雅罗鱼亲缘关系较近,准噶尔雅罗鱼是3种雅罗鱼中较古老的鱼种。     

新疆三种雅罗鱼属鱼类mtDNA D-loop多态性及起源分化分析

用PCR-RFLP技术,对新疆分布的准噶尔雅罗鱼、贝加尔雅罗鱼和高体雅罗鱼的mtDNA D-loop高变区约827bp进行了扩增,用ScaI、HinfI、AluI、DdeI 4种限制性内切核酸酶对56个样本的扩增产物酶切和RFLP分析,共检测到6种单倍型。准噶尔雅罗鱼存在2种单倍型：BDAA和BDBA;贝加尔雅罗鱼存在3种：AAAA、ABAA和ACAA;高体雅罗鱼只有1种。初步认为,准噶尔雅罗鱼、贝加尔雅罗鱼存在较丰富的群体内变异。用6种单倍型及净遗传距离绘制的UPGMA分子系统树,提示准噶尔雅罗鱼有可能是原始种,贝加尔雅罗鱼和高体雅罗鱼是由准噶尔雅罗鱼进化而来。 Abstract:PCR-RFLP technique was employed to amplify about 827bp of mtDNA D-loop hypervariable region of Leuciscus baicalensis,L.merzbacheri and L.idus in Xinjiang.The PCR products of 56 samples with four restriction enzymes were digested:ScaI,HinfI,AluI,DdeI,and RFLP analysis was done then.The study indicates that all of L. species and populations have six haplotypes,L.merzbacheri has two haplotypes:BDAA,BDBA;L.baicalensis has three:AAAA,ABAA,ACAA;L.idus has one:CAAA.It is primarily considered that L.merzbacheri and L.baicalensis have more intra-population mutations.The UPGMA trees with 6 haplotypes and net genetics distance pointed out that L.merzbacheri might be original species,L.baicalensis and L.idus are evolved from L.merzbacheri.     

准噶尔雅罗鱼染色体核型及带型的初步研究

以肾细胞作材料,采用秋水仙素-低渗-空气干燥法、Ag-NORs、C-带和G-带显带技术对准噶尔雅罗鱼(Leuciscus merzbacheri)染色体进行了研究。结果表明:(1)准噶尔雅罗鱼2n=50,核型组成为18m+14sm+6st+12t,NF=82,没有异型性染色体分化。(2)Ag-NORs的数目在不同的细胞中表现出多态性,数目为1～2个,出现1个Ag-NORs的频率最低(10%),出现2个的频率最高(70%);Ag-NORs主要出现在m1对和m4对同源染色体上;未发现有Ag-NORs联合的现象。(3)准噶尔雅罗鱼的染色体均呈现C-带阳性,可分为着丝粒C-带和端粒C-带。(4)同源染色体上G-带带纹基本一致,其带纹在每对染色体上的数目及分布具有明显特征性。     

准噶尔雅罗鱼β-肌动蛋白基因启动子克隆及序列分析

利用PCR方法克隆了准噶尔雅罗鱼(Leuciscus merzbacheri)的β-actin基因启动子片段SZ21,大小是2398bp。对克隆的启动子序列进行了转录调控元件的生物信息学预测分析,同时,基于启动子中包含的开放阅读框和内含子序列,探讨了准噶尔雅罗鱼与鲤鱼(Cyprinus carpio)、草鱼(Ctenopharyngodon idella)、青鱼(Mylopharyngodon piceus)、团头鲂(Megalobrama amblycephala)、泥鳅(Misgurnus mizolepis)间的系统进化关系。结果显示,该启动子序列的3个核心启动子转录元件:CAAT-box、CArGmotif和TATA-box分别在转录起始位点(+1)上游的-89、-59、-26处,序列中还含有MEF2、SATB、CHRF、INRE、MTEN、E-box、RU49、ZBPF、CREB、Enhance region、CEBP位点等多种转录调控元件。在剪接体内含子中,剪接位点遵循GT…AG法则。启动子SZ21序列含有3个内含子和155个氨基酸。内含子1、内含子2、内含子3的系统发育分析表明,团头鲂与草鱼和青鱼的亲缘关系要比与准噶尔雅罗鱼的更近一些,这与传统分类中的亲缘关系显示不一致,其原因尚需探讨。     

两种鯻科鱼类线粒体16S rRNA基因片段序列分析

利用PCR技术成功扩增了高体革鯻(Scortum barcoo)和厚唇弱棘喇(Hephaestus fuliginosus)的线粒体16S rRNA基因序列.通过序列测定,得到791 bp的基因片段,碱基A、T、G、C平均含量分别为31.4％、21.2％、20.5％和26.9％,序列中的A+T含量明显高于G+C的含量,这与其他鱼类的16S rRNA基因片段研究结果相一致.通过序列分析发现,高体革鯻和厚唇弱棘喇两序列共存在23处碱基变异,其中碱基转换位点20个,碱基颠换位点3个,转换与颠换比率为6.7,没有发现碱基插入与缺失.与从GenBank中查到的3种蜊科鱼类的同源序列比对,邻接法(neighbor-joining)构建亲缘关系树,结果显示,5种蜊科鱼类聚在一起,分为独立的2支,匀蜊和单色匀鯻及花身蜊聚成1支,高体革鯻和厚唇弱棘鯻聚为1支,说明高体革鯻与厚唇弱棘蜊的亲缘关系比较近,而与其他另外3种鯻科鱼类亲缘关系比较远.     

准噶尔雅罗鱼β-肌动蛋白基因启动子的克隆及其活性功能初步检测

以开发利用新疆濒危鱼类准噶尔雅罗鱼(Leuciscus merzbacheri)基因资源为研究目的, 利用PCR技术克隆准噶尔雅罗鱼的β-actin 基因, 得到的β-actin 基因片段SZ21包含启动调控区, 大小为2 398 bp。SZ21的启动调控区包括β-actin 基因上游调控序列、第1、第2、第3和第4外显子部分序列。上游调控序列中含有对转录起重要作用的CAAT框、TATA 框和CArG 框等元件。对启动子序列在线分析表明, 获得的启动子含有E-box、RU49、ZBPF、CEBP、CREB等多个重要转录因子结合位点。用AatⅡ破坏真核表达载体pEGFP-N1-AFPⅢ中的CMV启动子, 将准噶尔雅罗鱼的SZ21启动调控区克隆到载体pEGFP-N1-AFPⅢ(CMV坏)上, 构建成重组表达载体β2 pEGFP-N1-AFPⅢ。脂质体转染BHK-21细胞。结果表明, 克隆的准噶尔雅罗鱼β-actin基因启动子SZ21具有启动EGFP报告基因在哺乳动物细胞中表达的活性。通过BHK-21绿色荧光细胞的传代证实, 克隆的启动子具有持续启动蛋白基因表达的活性, 在细胞传代中可以遗传。PCR检测传代的BHK-21绿色荧光细胞基因组DNA, 均能检测到SZ21目的片段。文章成功分离了具有活性功能的准噶尔雅罗鱼β-actin基因启动子。     

从1 2S rRNA基因序列探讨8种鳄类的系统学关系

测得扬子鳄（Alligator sinensis）和暹罗鳄（Crocodylus siamensis）,的mtDNA12SrRNA基因片段的部分序列,与GenBank中的2种鳄及文献中4种鳄的12SrRNA基因相应片段,经比对后构建系统树。其结果显示,现存鳄类为单系起源,可划分为3个科,即：鳄科、食鱼鳄科和假食鱼鳄科。食鱼鳄与假食鱼鳄亲缘关系较近,支持将假食鱼鳄作为食鱼鳄科的一个属,与以形态学为基础的研究结果不同。在钝吻鳄科中,扬子鳄与凯门鳄亲缘关系较远,而与密西西比鳄的亲缘关系虽较近,但它们的确存在很多差异。两者12SrRNA基因序列差异达12．12％,碱基的颠换数为9。在基于碱基转换和颠换的NJ系统树及仅基于碱基颠换的NJ系统树中,前者支持扬子鳄与密河鳄间的亲缘关系较近,而后者支持扬子鳄与凯门鳄间的亲缘关系较近,说明扬子鳄与密河鳄间亲缘关系是值得研究的问题。因本文仅根据12SrRNA基因部分序列来进行分析的,尚需进一步研究。     

基于线粒体Cyt b序列对新疆额尔齐斯河贝加尔雅罗鱼遗传结构的分析

采用线粒体Cyt b基因序列对额尔齐斯河流域的青河(QH,18尾)、哈巴河(HBH,21尾)、185团(185T,18尾)和乌伦古湖(WLG,20尾)4个贝加尔雅罗鱼(Leuciscus leuciscus baicalensis)群体进行了遗传结构的比较分析。在片段长度为1 109 bp的同源序列上,77尾个体共检测到54种单倍型,其中共享单倍型7个,总单倍型多样性指数(Hd)、总核苷酸多样性指数(π)和平均核苷酸差异数(K)分别为0.981 9、0.008 21和9.091,且185团群体单倍型多样性指数和核苷酸多样性指数最高、青河群体最低。4个群体间遗传距离在0.006~0.011之间,基于Kimura 2-parameter法构建的单倍型邻接关系树分为3支,群体间遗传关系和地理分布没有明显的相关性。AMOVA分析显示额尔齐斯河流域贝加尔雅罗鱼遗传差异极显著(P0.01)。青河和乌伦古湖群体基因流(Nm)远高于其他群体间,推测乌伦古河或许是二者进行基因交流的渠道。尽管单倍型核苷酸不配对分布呈双峰,但偏差平方和(Q)与粗糙指数(r)均不显著(P0.05),且Tajima′s D和Fu′s Fs检验也均显著偏离中性,结合群体呈现高单倍型多样性和低核苷酸多样性的特点,推测贝加尔雅罗鱼经历了群体扩张事件。参考已校正的鲤科鱼类Cyt b基因0.76%/Ma的进化速率,估算群体扩张的时间大约在1.97 Ma前的更新世中晚期,推测该时期阿尔泰山地区发生的冰川作用和频繁的古地震造成的地理隔离和融合可能是贝加尔雅罗鱼发生群体扩张的重要原因。     

松江鲈鱼Cytb基因序列分析及种质鉴定

采用PCR方法扩增四尾松江鲈鱼线粒体细胞色素b(mitochondrial cytochrome b,Cytb)基因序列,全序列长度为1 141 bp,并建立了松江鲈鱼的实时定量PCR检测方法对该物种进行种质鉴定。分析Cytb基因序列基本特征显示,Cytb基因在第3位密码子表现出明显的反G偏倚,显示出脊椎动物Cytb基因的共同特性。第2位密码子嘧啶的含量远高于嘌呤的含量,有5个位点发生转换,0个位点发生颠换。选取杜父鱼科(Cottidae)的10种鱼类,与松江鲈鱼进行序列分析,构建发育系统树并分析遗传距离,与鲈鱼的亲缘关系最远。以松江鲈鱼Cytb基因序列为靶基因,利用PRIMER EXPRESS2.0软件设计引物和Taqman探针,建立检测松江鲈鱼的定量PCR方法,特异性强,灵敏度高,检测限为3.20×102拷贝/反应。     

额尔齐斯河高体雅罗鱼指环虫种群动态及中国一新记录种（英文）

2009年4月到2012年7月对额尔齐斯河(中国段)高体雅罗鱼鳃部寄生的指环虫进行了取样调查,以期了解该鱼指环虫的种群生态学特点。结果表明,高体雅罗鱼共感染了三种指环虫,分别为号筒指环虫、小刺指环虫、大翼指环虫,该鱼的总感染率为28.47%,平均感染强度为7.39(1—42)。感染率、感染强度在不同体长组的宿主中表现出不同的变化趋势,指环虫的感染率在30 cm以上的宿主中达到最大,为50%,而在L10范围内未发现感染;在不同体长组宿主中指环虫种群的分布类型主要为聚集分布,另外,根据形态学特征鉴定大翼指环虫(Dactylogyrus alatus f.major Linsyow,1878)为我国指环虫属一新记录种,论文对其主要形态特征进行了描述并绘制了特征图,同时与寄生于高体雅罗鱼鳃上的国内已记述指环虫属虫种的主要形态特征作了比较。     

Effects of repeated blood sampling on some blood parameters in freshwater fish

The effects of repeated weekly blood sampling on blood parameters were studied over a period of 22 days in a salmonid, Salmo gairdneri , and cyprinid fish, Leuciscus idus . The procedure induced mild anaemia demonstrated by decrease of red blood cell counts, haemoglobin and PCV. The changes were more marked in L. idus , which showed signs of increased red cell destruction. No significant trends could be observed in the white blood picture. The results show that blood tests are possible in individual fish, especially S. gairdneri over a long period.     

Factors influencing movement behaviour and home range size in ide Leuciscus idus

Seventeen individuals of ide Leuciscus idus were radio-tracked weekly from September 2003 to September 2004 in the River Elbe, Czech Republic, to examine migration patterns and the influence of environmental factors on their diurnal behaviour. Of the 10 environmental factors measured, L. idus were significantly influenced by turbidity, which increased diurnal movement and the home range size of the species. The peak of longitudinal movement occurred in the spring, indicating pre-spawning migration. Migrating fish moved downstream and later returned upstream to the vicinity of their original locations, displaying a homing behaviour.     

Heterogeneous colonization pattern of European Cyprinids, as highlighted by the dace complex (Teleostei: Cyprinidae)

The dace (Leuciscus leuciscus), with a very large geographic distribution all over Europe, represents an interesting species model for studies of the global mechanisms underlying aquatic system biodiversity. To assess the congruence with the past colonization process hypothesis of the freshwater fauna in Western Europe, we investigated the evolutionary history of this species, by integrating morphological variation (eight meristic characters), mitochondrial (cytochrome b, 16S rDNA and control region, over a total of 2169 bp) and nuclear (12 allozymes loci) phylogenetic relationships, and investigating population dynamics via expansion, migration, bottleneck, and divergence time analyses. We carried out nested clade phylogeographic analysis for a total of 663 specimens from 31 populations taken from all over the distribution area. Unlike previous studies, we found that L. leuciscus is currently constituted by five lineages belonging to two clades (yielding 6.3% of pairwise divergence). The relationships between these lineages were accounted for by complex biogeographical patterns due to Pliocene and Pleistocene paleoclimatic events, validating the identification of new glacial refuges for freshwater fish in Western Europe. Finally, we demonstrated hybridization between L. leuciscus and Leuciscus idus.     

A comparative study of neuropeptides in the intestine of two stomachless teleosts (Poecilia reticulata,Leuciscus idus melanotus) under conditions of feeding and starvation

Summary Endocrine cells containing bombesin-, enkephalin-, gastrin/CCK-, 5-HT-, and substance P-like material were demonstrated in the alimentary tract of Poecilia reticulata and Leuciscus idus melanotus. Endocrine cells with neuropeptide-Y-like immunoreactivity were found only in P. reticulata, those with VIP-like immunoreactivity only in L. idus melanotus. Gut nerves showing bombesin-, G/CCK-5-HT-, neurotensin-, substance P-and VIP-like immunoreactivity were observed in both species investigated, enkephalin- and neuropeptide Y-like immunoreactivity in P. reticulata alone. The distribution and amount of endocrine cells and nerves along the gut as visualized with the appropriate antisera varied in both teleosts. Histologically, the intestinal tract of these stomachless fish can be divided into three regions. A large number of endocrine cells with VIP-like immunoreactivity was noted in the rectum of L. idus melanotus. Endocrine cells containing bombesin-, enkepha-lin- and substance P-like material were found only in intestinal parts I and II in L. idus melanotus. Neuropeptide Y-like immunoreactivity was absent from intestinal part I of P. reticulata. The influence of starvation on the immunoreactivity of nerves and enteroendocrine cells in the teleost intestine was examined. After a starvation period of more than 6 weeks, no alterations were observed either in the appearance or amount of nerve and endocrine cell immunoreactivity.     

华南西部及海南岛美丽小条鳅种群遗传变异与亲缘地理

通过分析108尾采自华南西部12条水系的美丽小条鳅(Micronemacheilus pulcher)mtDNA 细胞色素b (cyt b) 基因全序列,研究其种群遗传变异和亲缘生物地理格局.美丽小条鳅的cyt b序列包含138个核苷酸变异位点(占全序列12.11%).分子变异分析(AMOVA)显示,分子遗传变异主要来自种群内(58.53%)和地理区内种群间(42.84%).采用邻接法构建的39个单倍型的NJ系统树显示,12条水系的美丽小条鳅聚成两支.其中,广西沿海诸独立水系(防城河、峒中河、北仑河、南流江)和横穿广东和广西的西江水系与广东漠阳江和潭江水系关系密切;而海南岛万泉河和南渡江与广东鉴江水系关系密切. 根据嵌套进化枝系地理分析(NCPA)推测,与越南毗邻的防城河周边地区可能是美丽小条鳅的扩散中心,该物种可由此区域通过两条途径扩散:1)沿西江水系向广西沿海独立水系至广东漠阳江和潭江水系扩散;2)向海南岛诸水系再至雷州半岛的鉴江水系扩散.在演化过程中,曾发生片断化事件、长距离建群和持续的分布区扩张.     

Experimental study of young fish distribution and behaviour under combined influence of baro-, photo- and thermo-gradients

The complex influence of baro-, photo- and thermo-gradients on distribution and behaviour of young physostomous Leuciscus leuciscus L., leaciscus idus L. and physoclistous fish Perca fluviatilis L. was investigated. 40 different combinations consisting of 4 types of photogradients, 3 types of termo gradients and 4 types of baro-gradients were tested. All considered factors influenced the distribution of physostomous and physoclistous fish with high degree of significance. Under a multi-factorial experiment fish behaviour and distribution are determined not only by a separately taken environmental factor but by the presence of other accompanying factors. Under different combinations of imposed factors, the reaction of fish to an individual factor and the character of the response (positive or negative) may change. Hydrostatic pressure is one of the strongest factors which influence fish distribution. The presence of its gradient may change the character of response of perch Perca fluviatilis to light (a changing of a sign of its photoreaction). Water temperature influences fish distribution more than illumination. However, the definite value of water temperature at which most fish prefer to stay may be changed depending on both the intensity of illumination and the value of hydrostatic pressure.     

6个中国猪地方品种和3个瑞典猪DNA分子系统发育相关关系

线粒体DNA遗传多样性用于评价6个中国地方猪种和3个瑞典家猪系统发育关系。采用PCR和序列分析方法得到了来自9个品种140头猪的线粒体中控制区440bp和细胞色素b基因798bp核苷酸序列。系统发育分析结果表明：6个中国地方猪种起源于亚洲野猪。中国地方猪种和欧洲野猪的线粒体DNA核苷酸序列变异发生在413000－875000年以前,而亚洲紧猪的变异仅发生在7000－156000上以前,由于2000年以前或18世纪初中国猪种导入欧洲家猪,因此瑞典家猪既属于欧洲类也属于亚洲类。