海带雌配子体细菌人工染色体(BAC)文库构建及特异分子标记邻近基因的图位克隆与序列

UV性别决定系统在一些低等生物生活史的单倍体阶段展现出特定的进化和遗传特性。实验构建了一个海带雌配子体基因组的细菌人工染色体(BAC)文库,结果显示,该文库包含31 872个克隆,插入片段平均长为115 kb,覆盖6.57倍的海带基因组。利用海带雌配子体特异性的标记FRML-1488的序列为探针筛选BAC文库,获得4个阳性BAC克隆。随机挑选一个克隆(638-C12),通过Roche454第二代测序平台进行测序,并经过间隔序列的扩增、克隆和测序,了解到该BAC克隆的插入片段长为86 996 bp。该序列位于海带基因组的Scaffold285上,占后者序列总长的22.4%。序列分析结果显示,在雌配子体特异性标记FRML-1488的上游存在大量的微卫星以及Copia逆转录转座子等重复序列。推测BAC克隆638-C12的插入片段可能与海带U染色体的性别决定区相关。本研究是BAC文库在海带性别相关序列图位克隆的首次应用报道,将有助于海带性别染色体结构的揭示及性别决定机制的解析。     

RAPD技术分析荷包红鲤抗寒品系与亲本的基因组变化

用４０个随机引物对荷包红鲤、荷包红鲤抗寒品系和黑龙江野鲤三个品系鱼进行ＲＡＰＤ反应,共扩增出３６６条带,获得多态性的ＤＮＡ片段６０条。荷包红鲤抗寒品系与荷包红鲤、荷包红鲤抗寒品系与黑龙江野鲤的基因组变异指数分别为０．７７和０．９０。说明荷包红鲤抗寒品系的基因组与其亲本黑龙江野鲤的基因组较荷包红鲤具有更大的变异。     

尼罗罗非鱼微阵列Fosmid基因组文库的构建及基因筛选

以pCC2FOS为载体构建尼罗罗非鱼Fosmid基因组文库,共挑选115 200个单菌落,保存在300块384孔样品板中,构建4 800个行池、7 200个列池、300个板池和25个超级池,形成微阵列,并对其进行复制备份。该文库插入片段平均长度约为40 kb,覆盖约罗非鱼基因组的3倍。连续传代实验研究表明,文库具有良好的稳定性。由于构建超级池－板池－行、列池三级池系统形成的微阵列,有助于快速、准确、有效地筛选目的基因,仅需77个PCR反应(超级池25 板池12 行池16 列池24)就能筛选到含有目的基因的一个单克隆,解决了普遍存在的文库筛选难题。通过文库尝试筛选18个与性别分化和生长相关基因,均获得2~5个阳性克隆,说明文库具有较好的实用性。     

条斑紫菜基因组Fosmid文库构建

高分子量基因组文库是开展条斑紫菜基因组学研究的必备工具。构建了由23 040个Fosmid克隆组成的条斑紫菜孢子体无偏倚基因组文库。检测分析表明：文库克隆重组率为100%；插入DNA片段长度为28~40 kb，平均长度为35 kb，文库覆盖率约为条斑紫菜基因组的2.78倍；从超级池中筛查条斑紫菜18S rRNA、atpA、h2A、rbcL基因，至少能得到一个阳性克隆，印证了计算所推测的文库覆盖率；随机选取6个Fosmid克隆经100代传代后插入DNA片段未发生丢失或长度变化，表明克隆传代的稳定性。该文库的构建为开展条斑紫菜基因组特性分析和基因克隆奠定了基础。     

鲤脑组织cDNA文库的构建及脑室管膜素基因鉴定

用Gubler-Hoffman法构建了低温下鲤(Cyprinus carpio Linnaeus)脑组织cDNA文库,经测定库容量为5.7×105 CFU/mL,文库的重组率接近95%,平均插入片段长度约为1.5 kb,符合文库保存和筛选实验的要求.同时,根据鲤与低温相关消减cDNA文库中低温下特异表达基因片段序列,设计PCR引物在此cDNA文库中进行PCR检测和序列测定,使用BLAST软件将测序的10个cDNA序列同GenBank等数据库进行比对,结果显示8个阳性克隆有相关同源性,2个克隆为功能未知的基因,全长率近75%;以上结果说明本实验所构建的cDNA文库质量较高,可作为筛选与鲤低温性状相关的功能基因的重要资源.同时对其中一个与低温相关的基因脑室管膜素基因构建了表达载体,为进一步验证该基因在鱼类低温适应中所发挥的作用及其作用机理奠定了基础.     

虾夷马粪海胆性腺cDNA文库构建及部分EST序列分析

应用Gubler-Hoffman cDNA文库技术.构建虾夷马粪海胆(Strongylocentrotus intermedius)性腺cDNA文库.测试结果表明.其库容量为2.10x 106 PFU/mL.长度范围在0.54.2 kb.插人片段平均长度为1.4 kb.达到建库要求.对虾夷马粪海胆cDNA克隆测序.将获得的819条EST序列与NCB1数据库进行BLAST比对.获得了65个有研究价值的EST序列和cDN^克隆.其EST序列已提交到GenBank.序列号分别为G0448010-GO448016,GR410172-GR410229.虾夷马粪海胆性腺cDNA文库的成功构建.使短期内获得大量调控海胆性腺生长和营养性状的关键基因表达信息成为可能.为进一步开发海胆的生物资源提供参考.     

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。     

剑尾鱼微卫星DNA的筛选

以剑尾鱼(Xiphophorus helleri)为材料,经MboⅠ限制性内切酶消化基因组DNA后,选取500～2 000 bp的片段连接到经BamHⅠ酶切的pUC18载体上,转化大肠杆菌DH5α构建部分基因组文库.采用设计合成的(AC)7、(GT)7重复序列为引物,PCR筛选部分基因组文库,对其中9个重组阳性克隆进行测序,结果共获得24个微卫星序列,其中Perfect(完美型)13个,占54.2%;Imperfect(非完美型)3个,占12.5%;Compound(混合型)8个,占33.3%.表明(AC/GT)n在剑尾鱼的基因组DNA中含量非常丰富.同时,根据其中3个克隆微卫星的侧翼序列设计引物,PCR扩增剑尾鱼基因组DNA,结果均扩增到目的片段.而且,这3对引物扩增出来的微卫星片段在非选育的剑尾鱼中显示出多态性,而在近交系19代则表现为单态,为剑尾鱼的实验动物化遗传研究提供了理论依据.     

绿色巴夫藻cDNA文库的构建及EST序列分析

将绿色巴夫藻(Pavlova viridis)培养液逐种加入氨苄青霉素、硫酸庆大霉素、硫酸阿米卡星3种抗生素处理,经检验后无杂菌污染。以该藻种为实验材料,以λTriplEx2为载体,利用SMART技术构建其cDNA文库。经测定文库滴度为6×106pfu/mL,重组率为98%。利用PCR方法检测cDNA文库插入DNA片段的大小,其长度为0.5~2 kb,表明该文库达到建库要求,可用来筛选低丰度mRNA的基因克隆。对cDNA文库中的阳性克隆进行随机PCR扩增,挑选插入DNA片段较大的克隆,进行5’末端单向序列测定,测序结果与NCBI数据库进行BLAST比对,获得了200个有研究价值的EST序列和cDNA克隆,为进一步研究和开发绿色巴夫藻的功能基因奠定了基础。     

黑龙江野鲤细菌人工染色体基因组文库构建

为了开展鲤基因组研究，深入研究遗传连锁图谱遗传标记的定位及数量性状的定位和克隆，并最终为分子育种提供服务，本实验构建了鲤的Cyprinus caxpio haematopterus）全血细胞制备琼脂糖凝胶包埋块的方法获     

Food and feeding habits of Tilapia aurea (Steindachner) (Cichlidae) in Lake Kinneret (Israel)

An analysis of the food of 274 specimens of Tilapia aurea (Steindachner), which had been collected in Lake Kinneret, showed that this species is mainly a zooplankton consumer.A study of the quantitative dynamics of the food components in the course of a year has proved that the species in question feeds more intensely in spring, this being the time when zooplankton forms are more abundant in the water of the lake. Vegetable detritus, mixed with plankton and benthos forms, served as additional and alternative food.The lack of clearly positive degree of food component selectivity, as well as the low values of the intestine repletion index and of the condition coefficient, account for the slowness in growth of Tilapia aurea in Lake Kinneret.The low values of the growth and feeding indices are due, according to the authors, to the pressure brought to bear by the other local fish species, which are competitors for the same food.     

Food and feeding habits of Tilapia zillii (Gervais) (Cichlidae) in Lake Kinneret (Israel)

Analysis of the gut content of 329 specimens of T. zillii (Cichlidae) collected from Lake Kinneret, has shown great variation in the sorts of food.A study of seasonal dynamics has proved the prevalence in the food of Chironomida pupae (Diptera) in winter and spring and of zooplankton forms in summer and autumn. The additional food consisted of the various groups of algae, the most frequently found being Cyanophyta (100%) and Pyrrophyta (64.16%). An extremely voraceous species, it consumes — while searching for its preferred food — anything that comes its way in the water: algae, scraps of macrophytes, autochthonous and allochthonous insects, and forms of benthic origin, such as Nematoda, Ostracoda, Porifera and Chironomida (larvae).The satiation index is high (between 4.08 and 5.63), in contradiction to the low values of the coefficient of condition (between 3.05 and 3.51), and with the slow rate of growth in Lake Kinneret. The main food of Tilapia zillii, consists of arthropod species with a chitin content (which is eliminated unchanged) of more than 50% of the total weight, and this may account for the poor exploitation of the trophic base.This species of fish may be considered as being detrimental to others (i.e. commercially important species) because of its successful competition for food, and not, as is often incorrectly assumed, because it is an aggressive consumer of their spawn and fry.     

叶尔羌高原鳅胚胎发育与胚后发育观察

采用形态学和生态学方法,对叶尔羌高原鳅[Triplophysa(Hedinichthys)yarkandensis(Day)]胚胎发育和胚后发育阶段全过程进行观察、拍照并测量。结果显示:叶尔羌高原鳅,卵微黏性,略有沉性,受精卵呈卵圆形,卵径为(0.60±0.052)mm,在水温(20.0±1.0)℃下,历时65 h 34 min完成整个胚胎发育分为7个生理阶段过程;胚后发育主要根据卵黄囊、体色、鼓鳔和须的变化分为仔鱼期、稚鱼期、幼鱼期。初孵卵黄囊仔鱼全长(2.0±0.65)mm,出膜后7 d,卵黄囊吸收完毕,完全消失;初孵仔鱼继续培育至16日龄,仔鱼鳃盖后缘鼓鳔明显长出,须清晰可辨,体色加深,心脏红色素明显,体色与成体相似,标志后期仔鱼发育完全进入稚鱼期,此时鱼苗全长(8.0±0.45)mm;培育至30日龄,仔鱼鼓鳔完全,鳃盖张合明显,身体透明特征消失,稚鱼阶段完成发育进入幼鱼期,此时全长达(13.0±0.55)mm,其外部形态和生态习性均与成鱼相似。试验中,卵黄囊长度(LY)和出膜天数(D)的关系式:LY=0.0286D2–0.0636D+3.1196(R2=0.9050);用直线方程拟合卵黄囊长度(LY)和卵黄囊仔鱼全长(LT)的关系式:LY=–1.315LT+5.368(R2=0.8199);拟合卵黄囊仔鱼全长(LT)和出膜后仔稚鱼天数(D)的关系式:LT=–0.0263D2+0.5113D+1.6169(R2=0.9890)。本研究旨在通过了解叶尔羌高原鳅的早期发育特征为该物种的保护和增殖对策提供科学依据,并对其苗种生产提供理论指导。     

Extinction of Daphnia lumholtzi (Sars) in Lake Kinneret (Israel)

Daphnia lumholtzi (Sars) was found in Lake Kinneret until the late nineteen-fifties. Lake Kinneret was the northern-most limit of the distribution of this species. It is suggested that fingerlings of grey mullets and Sarotherodon aureus that were introduced into Lake Kinneret caused the extinction of D. lumholtzi.     

Substitution effect of sea tangle (ST) (Laminaria japonica) with tunic of sea squirt (SS) (Halocynthia roretzi) in diet on growth and carcass composition of juvenile abalone (Haliotis discus,Reeve 1846)

Substitution effect of sea tangle (ST) with tunic of sea squirt (SS) in diet on growth and carcass composition of juvenile abalone was determined. One thousand four hundred and seventy abalones were distributed into 21 containers. Six formulated diets in triplicate were prepared. A 200 g/kg ST was included into the ST0 diet. The 200, 400, 600, 800 and 1000 g/kg of ST were substituted with the same amount of tunic of SS, referred to as the ST200, ST400, ST600, ST800 and ST1000 diets, respectively. Finally, Undaria was prepared to compare effect of the formulated diets on performance of abalone. The experimental diets were fed to abalone for 16 weeks. Weight gain of abalone fed the ST400 diet was higher than that of abalone fed the ST0, ST600, ST800 and ST1000 diets and Undaria. Weight gain of abalone fed the formulated diets was higher than that of abalone fed the Undaria. The chemical composition of the carcass of abalone was affected by dietary substitution of ST with tunic of SS. In conclusion, ST could be completely substituted with tunic of SS without retardation in performance of abalone. Abalone fed the ST400 diet substituting 400 g/kg ST with tunic of SS achieved the best growth.     

褐牙鲆(♀)×犬齿牙鲆(♂)杂交F1及其亲本肌肉营养成分分析与比较

褐牙鲆(Paralichthys olivaceus)为东亚地区重要的海水养殖鱼类,在中国、日本和韩国海水养殖业中占有重要的地位.目前,褐牙鲆经过连续多代缺乏选择的人工繁育和养殖,造成养殖群体种质发生退化,生长速度和抗逆性下降,极大制约了牙鲆养殖业发展.以选育生长迅速、抗逆性强、饵料转化率高的牙鲆类养殖新品种为目的,作者对褐牙鲆(♀)×犬齿牙鲆(Paralichthys dentatus)(♂)的杂交育种进行了研究.本实验对褐牙鲆(♀)×犬齿牙鲆(♂)杂交F1及其亲本肌肉营养成分进行测定.结果显示,杂交F1含水率显著低于双亲(P＜0.05),粗蛋白含量显著高于双亲(P＜0.05),粗脂肪含量介于二者之间;杂交F1的氨基酸总量、必需氨基酸、非必需氨基酸、半必需氨基酸与鲜味氨基酸总量都显著高于褐牙鲆与犬齿牙鲆(P＜0.05);脂肪酸含量方面,脂肪酸总量、不饱和脂肪酸总量3种鱼由高到低排列顺序为褐牙鲆、犬齿牙鲆、杂交F1,三者饱和脂肪酸总量差异不显著(P＜0.05).对3种鱼肌肉的营养品质进行了评价,杂交F1的必需氨基酸指数EAAI(90.30)明显高于褐牙鲆(77.75)与犬齿牙鲆(79.34),在蛋白质品质上表现出一定杂种优势.上述结果表明,杂交F1的肌肉具有蛋白和氨基酸含量较高、脂肪酸含量较低的特点.     

Development of embryos in Mastacembelus mastacembelus (Bank & Solender, 1794) (Mesopotamian spiny eel) (Mastacembelidae)

In this study, the embryonic development of the eggs in the Mastacembelus mastacembelus (Bank & Solender, 1794) was examined. At the same time, possibilities of artificial breeding through artificial insemination were investigated. Artificial insemination was achieved by mixing the eggs of the mature female and sperm of the mature male samples caught with gill nets (22 × 22) in Ataturk Dam Lake in Turkey. To this end, first in a Petri dish (100 × 20), the testes were cut into small pieces with a lancet and the mixture of sperm–testes‐tissue was obtained. The fertilization rate of the eggs was found to be 80%. The diameter of the eggs ranged from 2.015to 1.147 mm. The perivitelline space formed 0.5 h after insemination. The first cleavage occurred at the animal pool 4 h after insemination. The oil droplets had fused to a single droplet 19 h after insemination. The blastoderm became an embryonic shield 30 h after insemination. The blastoderm covered almost half the egg 40 h after insemination and embryonic body was formed. The blastoderm covered almost the whole egg 50 h after insemination. Some somites were discernible 59 h after insemination. The embryonic body reached two‐third of the circumference of the egg 70 h after insemination. The tail bud began to separate from the yolk 77 h after insemination. A newly hatched larva was observed at 85 h after insemination.     

Complete developmental cycle of Myxobolus pseudodispar (Gorbunova) (Myxosporea: Myxobolidae)

Myxobolus pseudodispar (Gorbunova) is a common parasite of the muscle of roach, Rutilus rutilus L., whereas its actinosporean development occurs in two oligochaete alternate hosts. This paper reports the complete developmental cycle of this parasite in the oligochaete alternate host Tubifex tubifex and the roach. In laboratory experiments, parasite-free T. tubifex specimens were infected by myxospores of M. pseudodispar collected from roach in Lake Balaton. Parasite-free roach fingerlings were infected with floating triactinospores (TAMs) released from oligochaetes on day 69 after challenge. Young plasmodia and spores in roach were first recorded on day 80 post-exposure (p.e.). Myxospores collected from experimentally infected roach initiated a new development in T. tubifex and the resulting TAMs infected roach. No infection of roach resulted from feeding oligochaetes containing mature triactinospores.     

Use of Onion (Allium cepa) and Garlic (Allium sativum) Wastes for the Prevention of Fungal Disease (Saprolegnia parasitica) on Eggs of Rainbow Trout (Oncorhynchus mykiss)

This study was designed to prevent fungal disease (Saprolegnia parasitica) that occurs on rainbow trout eggs (Oncorhynchus mykiss) by using wastes of onion (Allium cepa) and garlic (Allium sativum) plants. For this purpose, fertilized rainbow trout eggs were exposed to garlic skin, garlic stem and onion skin aqueous methanolic extracts by bathing in concentrations of 0.4, 0.8, 1.6 and 3.2 g/L, whereas the control group was left untreated. The larvae in all groups were monitored until they become free-swimming larvae, the number of eggs died due to fungus was recorded, and the data obtained from experimental groups and control group were compared. As a result, it was determined that onion skin had no effect on the number of eggs that died due to fungus (p > .05), while garlic skin and garlic stem extracts significantly reduced the number of fungal infestations without affecting the number of live larvae or the embryological development of the eggs (p < .05). In light of these data, we conclude that garlic skin (0.4, 0.8, and 1.6 g/L) and garlic stem (0.8 and 1.6 g/L) aqueous methanolic extracts are effective at preventing Saprolegnia parasitica infestation on rainbow trout eggs and may be used in aquaculture.     

Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp

Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. 2013); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen‐free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV‐infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid‐body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve‐SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.