牙鲆基因组 (CAG)n微卫星 DNA 特征分析

通过磁珠富集法筛选牙鲆(Paralichthys olivaceus)的微卫星分子标记,采用限制性内切酶Sau 3A Ⅰ对牙鲆完整基因组DNA进行酶切;通过蔗糖溶液梯度离心,收集400～900 bp大小的片段,连接Brown接头,构建牙鲆基因组文库.用生物素标记的微卫星探针(CAG)15,对基因组文库进行杂交,利用磁珠富集含有微卫星的DNA单链序列,并对其进行PCR扩增;将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星序列文库.利用大量质粒检测法进行二次筛选,成功地从牙鲆基因组中分离出含有CAG重复的微卫星序列,测序其中的3000个单菌落,获得2805个(占93.5%)含有微卫星序列的克隆,其中含有微卫星座位3120个,完美型1808个,占57.97%;非完美型226个,占7.25%;混合型1085个,占34.78%.从中选出186个微卫星序列设计120对引物并合成,经过筛选,74对引物可扩增清晰条带,其中68对呈多态性.     

磁珠富集法制备大口鲶的微卫星分子标记

通过磁珠富集的方法分离大口鲶（Silurus meriaionalis）的微卫星分子标记。将基因组DNA酶切，梯度离心收集400～900bp片段并纯化，连接Brown接头，用生物素标记的寡核苷酸（CA）15作探针与其杂交，杂交复合物结合到包被有链霉亲和素的磁珠上，然后将这些片段洗脱，PCR扩增，进行克隆构建“基因组文库”，再通过同位素标记的探针（CA）15进行2次杂交筛选，所得到的阳性克隆测序。从所获得593个阳性克隆中选取178个经测序，97．19％（173个）含有微卫星序列，90．60％重复数在10以上，75．98％为完美型。除探针中使用的CA重复单元外，还观察到Cr、GA、ATG的重复序列。设计获得120对微卫星引物，合成40对经PCR筛选，结果31对引物扩增出多态性条带。显示出，磁珠富集法是获得微卫星分子标记的一种有效的方法，可成为今后发展该标记的主要方法。同时，制备出的微卫星标记可为今后研究大口鲶的分子遗传育种提供有用的遗传标记。     

磁珠富集法分离草鱼微卫星分子标记

磁珠富集法是一种快速、高效的分离微卫星分子标记的方法。本研究通过该方法分离草鱼的微卫星分子标记。将草鱼（Ctenopharyngodon idella）基因组DNA经Sau3AI酶切，同收纯化400～900bp片段，连上接头，构建“基因组PCR文库”。用生物素标记的简单重复序列（CA）15作探针与其杂交，杂交复合物结合到包被有链霉亲和素的磁珠上，经一系列的洗涤过程，去除磁珠表面不含有微卫星的片段。将吸附在磁珠上的片段洗脱，PCR扩增放大，再进行克隆和测序，根据微卫星两端的保守序列设计引物，即可得到微卫星分子标记。本研究义通过同位素标记的探针（CA）15进行二次杂交筛选，获得阳性克隆132个，所得到的阳性克隆经测序，86．36％含有微卫星序列，共获得130个微卫星DNA序列。用引物设计软件Primer Premier5．0没计引物83对。     

合浦珠母贝微卫星DNA标记分离与分析

采用生物素标记的（CA）15、（AG）12、（ACA）15、（GATA）8、（GATT）75个探针和磁珠富集法构建马氏珠母贝（Pinctada martensii Dunker）基因组微卫星富集文库。随机挑选500个进行PCR筛选，得到138（27．6％）个候选克隆，测序分析发现130个克隆含有微卫星基重复单元。进一步通过序列比对，最终获得109个具有特异微卫星序列的阳性克隆，其中包含179个微卫星DNA结构域。获得的微卫星序列中，属于完全型序列的有154条，不完全型重复型序列有19条，复合型重复序列有6条。序列长度为70～490bp，平均295bp。微卫星核心序列两碱基重复3到39次，绝大多数序列重复次数大于10。基于微卫星两端的侧翼序列设计并获得了13对能够在马氏珠母贝基因组有效扩增的微卫星引物。本研究旨为进一步开展马氏珠母贝分子育种及资源评价分析提供基础资料。     

脊尾白虾微卫星富集文库的构建与多态性标记的筛选

采用人工合成的生物素标记(AG)15探针及磁珠富集法构建了脊尾白虾基因组微卫星富集文库.将脊尾白虾基因组DNA经HaeⅢ酶切后,收集400 ～1 200 bp片段,连接特定接头,与生物素标记(AG)15探针杂交并捕获含有微卫星的DNA片段,然后连接到pMD18-T载体中,构建脊尾白虾微卫星富集文库.随机挑取947个克隆,经菌落PCR检验,其中667个为阳性克隆.从阳性克隆中随机选取184个克隆进行测序,有150个克隆含有微卫星序列,共获得199个微卫星序列,其中完美型158个(79.4％),非完美型27个(13.6％),复合型14个(7.0％).根据微卫星序列,设计了119对微卫星引物,64对扩增出稳定的具有特异性的条带,经30个脊尾白虾个体进行多样性评价后,有26个位点表现出多态性.26个微卫星位点获得的等位基因数从3 ～ 16个不等,平均每个位点扩增得到6.5个等位基因.观测杂合度(Ho)、期望杂合度(4)及多态性信息含量值(PIC)的范围分别为0.111 ～ 0.929、0.246 ～0.932、0.231 ～0.909.本研究开发的微卫星位点将为脊尾白虾种群多样性研究、家系识别和种质鉴定提供有效的工具.     

哲罗鱼基因组微卫星富集文库的构建与分析

采用磁珠富集法构建哲罗鱼（Hucho taimen Pallas）荩因组微卫星文库。哲罗鱼基因组DNA经MboⅠ限制性内切酶消化后，选取400-900bp的片段，用生物素标记的简单重复序列（ACA）15作探针与其杂交，杂交复合物结合到包被有链霉亲和素的磁珠上，获得目的片段，连接T载体克隆，构建基因组微卫旱富集文库。再用同位素标记的（ACA）15探针进行二次筛选，筛选出686个阳性克隆，阳性克隆率为35．94％。对其中140个阳性克隆进行测序，共获得149个微卫星序列，4个小卫星序列，其GenBank Accession Number为DQ110955～DQ121108。其中perfect（完美型）62个，占41．61％；imperfect（非完美型）92个，占61．74％；compound（混合型）5个，占3．36％，重复次数主要分布于6～45（81．21％），平均重复次数为32．5，这表明（ACA／TGT）。在哲罗鱼基因组DNA中含最非常丰富。本研究旨为从DNA水平上研究哲岁鱼种群结构、遗传多样性及目前群体现状等方面提供有效工具。     

三角帆蚌微卫星富集文库的构建、鉴定及多态性分析

通过磁珠富集法构建了三角帆蚌(Hyriopsis cumingii)微卫星富集文库。共获得800个阳性克隆,其中的100个阳性克隆经测序分析,共获得微卫星序列54个。利用这些微卫星序列可设计33对引物,经过PCR扩增筛选获得了25对可用引物。利用聚丙烯酰胺凝胶电泳,对洞庭湖1个种群的30只三角帆蚌进行扩增,发现13对引物呈现多态性,等位基因数在4～9。观测杂合度在0.2543～0.9827,期望杂合度在0.3629～0.8217,信息多态含量平均为0.5198。由于存在无效等位基因,两个微卫星基因座(GenBank登录号为GQ302651和GQ302658)明显地偏离哈代-温伯格平衡,但没有发现连锁不平衡现象。这说明利用磁珠富集法构建的三角帆蚌微卫星文库质量较好,13对多态性微卫星引物可为三角帆蚌微卫星连锁图谱构建、分子进化和系统发育研究、分子标记辅助育种以及经济性状的QTL定位提供帮助。     

FIASCO法筛选鳜鱼微卫星标记

通过FIASCO(Fast Isolation by AFLP Sequences Containing repeats)法构建鳜鱼(Siniperca chuatsi)基因组微卫星富集文库,分离微卫星DNA序列并对其特征进行分析。鳜鱼基因组DNA经MseⅠ限制性内切酶消化后,选取200~800 bp的片段与MseⅠ接头连接,用生物素标记的寡核苷酸探针(AC)8、(CT)8、(AT)7、(GATA)8、(GATT)7与其杂交,杂交复合物结合到包被有链霉亲和素的磁珠上,变性洗脱获得单链目的片段,经PCR扩增形成双链,然后克隆到pGEM-T载体上,转化至DH5α中,首次成功构建鳜鱼基因组微卫星富集文库。对其中100个阳性克隆进行测序,60个(60%)含有微卫星序列(GenBank Accession Number:DQ789247~DQ789306)。成功设计了47对鳜鱼微卫星引物,并合成21对引物进行PCR扩增,结果筛选出18个多态性微卫星标记。结果表明:FIASCO法能有效提高筛选微卫星标记的效率。本研究筛选的微卫星标记可以用于鳜鱼遗传背景分析和遗传连锁图谱构建,并将为鳜鱼基因组结构分析、标记辅助育种以及数量性状位点(QTL)基因的定位等研究提供候选微卫星标记。     

青蟹微卫星DNA的筛选及特征分析

利用磁珠富集法,结合生物素标记的(CA)16寡核苷酸探针从青蟹基因组MboI酶切片段中筛选微卫星DNA序列.将得到的片段与pGEM-T Easy载体连接后转化克隆构建微卫星富集文库.经PCR筛选检测,对89个阳性克隆进行测序,其中52个克隆中含有64个微卫星,完全型占87.50%,重复次数超过11次的占78.12%.根据所获微卫星的侧翼序列设计并合成了30对引物,通过优化PCR反应条件,并在35只青蟹个体中进行PCR扩增检测,最终获得了10对具有多态性的引物,为进一步开展青蟹遗传多样性分析、遗传图谱构建等研究提供了基础资料.     

日本蟳微卫星富集文库的建立与多态性标记的筛选

采用磁珠富集法筛选日本蟳微卫星分子标记。日本蟳基因组DNA经Sau3 AⅠ酶切后,收集400~1 200 bp大小的片段并纯化,利用生物素标记的寡核苷酸探针(AC)¹⁵从中筛选出含有微卫星序列的DNA片段,连接到pMD18-T载体中,构建富集微卫星序列的基因组文库,经PCR检测筛选出阳性克隆进行测序。从随机挑选的970个菌落中筛选出369个阳性克隆进行测序,结果86.99%(321个)含有微卫星序列,其中完美型占80.54%,非完美型占15.95%,混合型占4.28%。除使用的探针AC重复外,还得到GA、CT等重复序列。共设计出102对微卫星引物,其中65对能扩增出清晰条带,27对具有多态性。同时筛选出的微卫星标记可为今后研究日本蟳的分子遗传育种提供有效的遗传标记。     

El Nino／La Nina影响下北部湾海域浮游桡足类种类组成与多样性

以1998年1月至1999年5月对北部湾海域进行的4个航次生态环境综合调查的资料为基础，分析El Nino／La Nina期间北部湾四季浮游桡足类种类组成与多样性，并对其与气候及海洋水文的关系进行初步探讨。结果显示，调查期间共出现浮游桡足类122种，其中32种周年出现，以春（82种）、夏（84种）较多，秋（52种）、冬（67种）较少；四季共出现优势桡足类13种，以广温广盐类型占主导，除微刺哲水蚤（Canthocalanus pauper）外，其余12种为季节性优势种；冬、夏两季受El Nino影响较大，优势种以中型桡足类为主；秋、春季大型桡足类优势地位较高，受La Nina影响明显；北部湾浮游桡足类多样性较高。分析表明，夏季北部湾水温和盐度对浮游桡足类群落多样性有一定影响，但在其他季节，这种影响不明显。     

Cell and tissue reactions of turbot Scophthalmus maximus (L.) to Tetramicra brevifilum gen. n., sp. n. (Microspora)

Abstract. Tetramicra brevifilum gen, n. sp, n. is described from the connective tissues throughout the body musculature of turbot Scophthalmus maximus (L.). Uninucleate spores (4.8·2 μm) are characterized by a large posterior vacuole, electron dense inclusion bodies in cytoplasm and nucleus and short filaments with 3–5 coils (50 μm extruded). Sporogony is apansporoblastic and tetrasporoblastic, within a host vacuole. Features of the xenoma (2 mm diameter) include a microvillous surface layer with microtubules and microfilaments and a reticulate nucleus with numerous nucleoli. Adherence between adjacent xenomas results in composite cysts of various sizes and shapes depending on site, being dendritic in the myomeres, arms extending between the fibres. Effects on the host, other than eventual destruction of the host cell, include displacement of muscle fibres in myomeres with loss of attachment to myocommata. Breakdown of the xenoma with release of spores into the connective tissue matrix was associated with a localized necrosis affecting surrounding muscle fibres, with leucocyte infiltration and collagen deposition. Spores phagocytosed by macrophages were destroyed in vacuoles, some with multi-laminate walls. Experimental infections established by intramuscular injection at marked sites remained localized, which suggested that xenomas afford sufficient scope for spore production without the need of secondary invasion. The swimming efficiency of turbot would be impaired in severe infections leading to lowered growth rates and increased mortality in wild populations due to predation and starvation. Feeding hierarchies exist in turbot and the disease could prove significant in farming conditions.     

Production of interploid triploids by 4n × 2n blunt snout bream (Megalobrama amblycephala. Yih) and their first performance data

Positive and negative interploid triploids of blunt snout bream (Megalobrama amblycephala) were produced by reciprocal crossing autotetraploids with diploids. Fertilization and hatching rate of negative interploid triploid, 2n♀× 4n♂, at different reproductive ages was similar to that of control, but higher than that of positive interploid triploid, 4n♀× 2n♂. Additionally, the development rate of embryos of the positive interploid triploid, 4n♀× 2n♂, was slower than that of control and the negative interploid triploid, 2n♀× 4n♂. Gonad growth of the positive and negative interploid 3n was significantly (P<0.01) affected by parents, and spawning could not be induced both in females and males at the 2+ age. The negative interploid 3n (2n♀× 4n♂) had a significantly higher survival rate than the positive interploid 3n (4n♀× 2n♂), but close to the level of the control (P<0.05). The daily growth rate of the negative interploid 3n was similar to that of the good breed ‘Pujiang No. 1’ blunt snout bream (2n) during the early life stages.     

Description and phylogeny of Ceratomyxa anko sp. n. and Zschokkella lophii sp. n. from the Japanese anglerfish, Lophius litulon (Jordan)

Two new species of myxozoans from the Japanese anglerfish, Lophius litulon, are described using myxospore morphology and small subunit rDNA sequences. Ceratomyxa anko sp. n. is a parasite of the gall bladder and had a prevalence of 57%. Mature spores of C. anko sp. n. are arcuate to crescent shaped with valves tapering to rounded tips. A prominent sutural line runs centrally between the round adjacent polar capsules containing the polar filament coiled two to three times. Spore measurements: length 10.8 (9.7-11.9) microm, width 41.9 (36.9-47.2) microm, polar capsule diameter 4.6 (4.1-5.3) microm. Ceratomyxa anko sp. n. can be distinguished from other Ceratomyxa spp. due to its spore dimensions and shape. Zschokkella lophii sp. n. is a parasite of the urinary bladder and had a prevalence of 70%. Mature spores are ellipsoidal to semicircular with bluntly pointed ends. The sutural line is curved or sinuous and the valves have no discernable surface ornamentation. Two almost spherical polar capsules are located separately in the ends of the spore, opening in almost opposite directions and contain the polar filament with five coils. Spore measurements: length 20.1 (16.8-24.0) microm, width 14.9 (12.7-16.8) microm, polar capsule diameter 5.1 (3.6-5.8) microm. Zschokkella lophii sp. n. can be distinguished from other Zschokkella spp. due to the terminal opening of the polar capsules within the spores and the site of infection within the host fish. In the phylogenetic analyses, C. anko sp. n. grouped with other members of the same genus forming a monophyly. Zschokkella lophii sp. n. forms a discrete clade with another Zschokkella sp. that infects the urinary bladder of marine fish. This grouping forms a sister clade to one containing members of the genus Parvicapsula, all of which are parasites of the urinary system in marine fish.     

Pentacapsula neurophila sp.n. (Multivalvulida) from the central nervous system of striped trumpeter,Latris lineata (Forster)

Striped trumpeter, Latris lineata, are being experimentally cultured by the Tasmanian Aquaculture and Fisheries Institute at Taroona, Tasmania. Fish surviving over 30 days post-hatching have frequently developed nervous aberrations associated with a severe granulomatous meningoencephalomyelitis. The myxozoan parasite Pentacapsula neurophila sp.n. was revealed as the parasite causing the disease in the striped trumpeter juveniles. Measurements made of isolated spores indicated that the organism was distinct from all previously described Pentacapsula species. This is the first report of a marine myxozoan parasite of the genus Pentacapsula in Australian waters.     

Effects of diets rich in linoleic (18:2n - 6) and α-linolenic (18:3n - 3) acids on the growth,lipid class and fatty acid compositions and eicosanoid production in juvenile turbot (Scophthalmus maximus L.)

Three practical-type diets utilizing fishmeal and casein as the protein sources and containing fish oil (FO), safflower oil (SO) or linseed oil (LO) were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 1.2 g for a period of 12 weeks. No differences in final weight, mortality or development of pathological lesions were evident either between duplicate tanks or between dietary treatments over this period. Fish fed diets containing SO and LO contained significantly greater amounts of liver triacylglycerol compared to fish fed FO. The major C₁₈ polyunsaturated fatty acids (PUFA) in SO and LO diets, 18:2(n-6) and 18:3(n-3) respectively, were readily incorporated into both total lipid and individual phospholipids of turbot tissues. There was no accumulation of the Δ6-desaturation products of these fatty acids, namely 18:3(n-6) and 18:4(n-3), in any of the tissues examined. The products of elongation of 18:2(n-6) and and 18:3(n-3), 20:2(n-6) and 20:3(n-3) respectively, accumulated in both total lipid and phospholipids with the highest levels of 20:2(n-6) in liver PC and 20:3(n-3) in liver PE. Eicosapentaenoic acid [EPA, 20:5(n-3)] levels exceeded those of arachidonic acid [AA, 20:4(n-6)] in phosphatidylinositol (PI) from liver and gill of fish fed LO. EPA levels in liver PI from fish fed LO were 3-fold and 2-fold greater than SO-fed and FO-fed fish, respectively. Fish fed diets containing SO and LO had significantly reduced levels of AA in liver and muscle total lipid and lower AA in individual phospholipid classes of liver and gill compared to FO-fed fish. The concentration of thromboxane B₂ was significantly reduced in plasma and isolated gill cells stimulated with calcium ionophore A23187 of fish fed SO and LO compared to those fed FO. Prostaglandin E produced by isolated gill cells stimulated with A23187 was significantly reduced in fish fed both SO and LO compared to fish fed FO.     

Myxobolus erythrophthalmi sp. n. and Myxobolus shaharomae sp. n. (Myxozoa: Myxobolidae) from the internal organs of rudd, Scardinius erythrophthalmus (L.), and bleak, Alburnus alburnus (L.)

During a survey of myxosporean parasites of cyprinid fish in Hungary, infections caused by unknown Myxobolus spp. were found in the internal organs of rudd, Scardinius erythrophthalmus, and bleak, Alburnus alburnus . Small plasmodia developed in blood vessels of the kidney, liver, testes and intestinal wall. The parasites were studied on the basis of spore morphology and by histological and molecular methods. In most cases, plasmodia were surrounded by host tissue without a host reaction; however, in advanced cases, a connective tissue capsule was seen around plasmodia. Spores collected from the two fish species differed from each other and from the known Myxobolus spp. both in their morphology and 18S rDNA sequences. The two species, described as M. erythrophthalmi sp. n. from rudd and M. shaharomae sp. n. from bleak, are characterized by a specific histotropism to blood vessels, while the organ specificity involves the kidney and for the latter species, most internal organs.     

Glugea heraldi n. sp. (Microsporida, Glugeidae) from the seahorse Hippocampus erectus Perry

Abstract. A new species of Microsporida, Glugea heraldi , is described from the seahorse Hippocampus erectus . Measurements of fixed spores and notes on the histopathology of parasitized tissue are given.     

Henneguya pagri n. sp. (Myxozoa: Myxosporea) causing cardiac henneguyosis in red sea bream, Pagrus major (Temminck & Schlegel)

Henneguya pagri n. sp. (Myxozoa: Myxosporea) is described from netpen-cultured red sea bream, Pagrus major, in Japan. Affected fish displayed anaemic gills, an enlarged bulbus arteriosus and internal haemorrhaging in the pericardial cavity. Disease outbreaks occurred during the summer and the prevalence of infection reached 75% in August. Plasmodia of H. pagri developed in the bulbus arteriosus of red sea bream, in which degenerative cardiomyopathy was observed. Following maturation of the parasite, the influx of spores into the gills caused congestion of the gill capillaries, resulting in proliferative branchitis. Spores (10.5 x 7.5 microm) were ovoid with two caudal appendages (29.6 microm in average length), with a whip-like extension from the end. Partial small subunit ribosomal RNA gene sequences of H. pagri are closely related to those of H. lateolabracis infecting the bulbus arteriosus of Chinese sea bass, Lateolabrax sp.