鲤鱼肌肉生长抑制素基因(MSTN)的克隆及其组织表达特征

肌肉生长抑制素(Myostatin,MSTN)是动物肌肉发育和生长过程中的负调控因子,对MSTN的研究将有助于促进动物生产。鲤鱼是我国的主要淡水养殖对象之一。因此,我们采用RT-PCR方法克隆了鲤鱼MSTN cDNA(No.EF551058)的部分序列,长度为921bp,编码306个氨基酸残基。鲤鱼MSTN具有MSTN的共同特征,有蛋白酶水解位点RIRR和9个保守的半胱氨酸残基。多重序列比较发现其与斑马鱼GDF8有极近的亲缘关系,96.7%的氨基酸序列同源。不同组织的RT-PCR分析发现鲤鱼MSTN主要在肌肉和脑部表达,而其他所检测组织未见表达。鲤鱼MSTN不仅在肌肉生长发育中发挥作用,可能在神经系统发育中也有其作用。     

The effect of high dietary zinc on trypsin activity in carp (Cyprinus carpio)

Carp fed pellets containing a range of zinc concentrations showed a significant increase in trypsin activity in the intestine with increasing zinc in the diet.     

A linkage map of common carp (Cyprinus carpio) based on AFLP and microsatellite markers

Common carp (Cyprinus carpio) is an important fish for aquaculture, but genomics of this species is still in its infancy. In this study, a linkage map of common carp based on Amplified Fragment Length Polymorphism (AFLP) and microsatellite (SSR) markers has been generated using gynogenetic haploids. Of 926 markers genotyped, 151 (149 AFLPs, two SSRs) were distorted and eliminated from the linkage analyses. A total of 699 AFLP and 20 microsatellite (SSR) markers were assigned to the map, which comprised 64 linkage groups and covered 5506.9 cM Kosambi, with an average interval distance of 7.66 cM Kosambi. The normality tests on interval map distances showed a non‐normal marker distribution. Visual inspection of the map distance distribution histogram showed a cluster of interval map distances on the left side of the chart, which suggested the occurrence of AFLP marker clusters. On the other hand, the lack of an obvious cluster on the right side showed that there were a few big gaps which need more markers to bridge. The correlation analysis showed a highly significant relatedness between the length of linkage group and the number of markers, indicating that the AFLP markers in this map were randomly distributed among different linkage groups. This study is helpful for research into the common carp genome and for further studies of genetics and marker‐assisted breeding in this species.     

烟酸诺氟沙星在松浦镜鲤体内的药动学特征

结合单纯聚集法和二步法,应用高效液相色谱(HPLC)技术研究了分别以10、30、60 mg/kg剂量对松浦镜鲤(Cyprinus carpio specularis)口灌烟酸诺氟沙星后,药物在实验鱼血浆中的药动学特征。3种给药剂量下,诺氟沙星在松浦镜鲤血浆中的血药浓度和时间关系均可用一级吸收二室开放模型进行描述,吸收半衰期(t1/2ka)分别为0.165、0.061、0.043 h,消除半衰期(t1/2β)分别为18.282、29.969、42.051 h,达峰时间(Tmax)分别为0.333、0.327、0.302 h,达峰浓度(Cmax)分别为4.780、6.247、12.689 mg/L,药时曲线下面积(AUC)分别为32.698、53.015、174.998 mg·h/L,表观分布容积(Vd)分别为1.044、4.347、4.561 L/kg。说明随着给药剂量的增加,诺氟沙星的吸收和消除速率均加快,给药剂量对药动学特征有显著影响。     

转反义sGnRH基因败育鲤鱼的性成熟诱导

采用反义转基因技术已成功获得整合CAsGnRHpc-antisense基因的转基因鲤鱼,简称AS-sGnRH F0鲤鱼.通过挤压1年龄AS-sGnRH F0鲤鱼的腹部,发现该群体中有精液/无精液个体比例为33/69,与对照组鲤鱼中有精液/无精液个体比例接近1：1相比存在显著差异.通过注射鲤鱼垂体抽提物诱导无精液AS-sGnRH F0鲤鱼恢复繁殖功能,获得3尾育性被恢复的AS-sGnRH F0鲤鱼.进一步的解剖观察发现,在无精液AS-sGnRH F0鲤鱼群体中,完全无性腺的AS-sGnRH F0鲤鱼比例约31.3%.根据直观的挤精液和解剖学观察,推测在AS-sGnRH F0鲤鱼群体中,雄鱼：雌鱼：＂脂肪型＂性腺个体的比例大约为35：45：20%.受精卵发育12 h后,统计A、B、C系AS-sGnRH F0鲤鱼精子的受精率分别为73.94、59.45和64.05%.从A、B、C系杂交后代F1分别随机取样100尾,PCR检测的阳性率分别为0、78.1和87.8%.研究结果表明通过补充外源激素可以使促性腺激素分泌不足而不育的AS-sGnRH F0鲤鱼恢复具有产生成熟配子的能力.     

Development of 10 tri- and tetranucleotide microsatellite loci for population studies in the common carp (Cyprinus carpio L.)

Ten tri- and tetranucleotide microsatellite DNA markers were isolated and characterized from common carp (Cyprinus carpio L.) to estimate genetic potential. These markers were tested in the samples from two closely related carp populations (Cyprinus carpio var. xingguonensis and Cyprinus carpio var. wananensis). The number of the alleles ranged from three to nine, and observed and expected hererozygosities varied from 0.207 to 1.000 and from 0.499 to 0.900 in each population, respectively. No evidence for linkage disequilibrium was found, indicating that these markers will be useful for population studies.     

锦鲤中吉氏库特菌的分离与鉴定

对从患病锦鲤(Cyprinus carpio L.)肝组织中分离的1株纯培养菌(HC050630C-1)进行了形态特征、主要理化特性、对健康鲤鱼的致病作用、药物敏感性等方面的检验;同时测定了该株菌的16S rRNA基因序列,构建了系统发育树。结果表明,被检菌为吉氏库特菌(Kurthia gibsonll),对健康鲤鱼未显示明显的致病作用;所测菌株的16S rRNA基因序列长度1459bp,在GenBank中登录号为EF611423;该菌株16SrRNA基因序列与GenBank数据库中吉氏库特菌的16S rRNA基因序列同源性在99%;药敏试验结果显示,对供试的氨苄青霉素等27种药物呈现高度敏感或敏感,对头孢吡肟等6种呈低度敏感,对苯唑青霉素等4种耐药。     

鲤鱼肝胰脏cDNA 文库的表达序列标签分析及Lb-Fabp mRNA的特征

本文构建了鲤鱼肝胰脏cDNA 文库,共获得了1016条有效的表达序列标签。拼接组装成115 个contigs和282 个singletons。其中215个拼接序列在GenBank公共数据库中寻找到相对应的基因。对它们进行功能性分类和比较分析为鲤鱼肝胰脏的研究提供了基因表达信息的基础。文库中1016条表达序列标签有11条代表了鲤鱼肝基本型脂肪酸结合蛋白(Lb-FABP)。通过序列比较我们获得了两个具有相同开放阅读框长度的Lb-Fabp cDNAs。开放阅读框全长381bp,编码126个氨基酸。半定量RT-PCR结合Southern blot技术研究了Lb-Fabp mRNA 在成鱼不同组织以及早期发育不同时期的表达图式。结果表明,Lb-Fabp mRNA 在肝胰脏、中肠和后肠中表达量较高。同时在精巢和皮肤中有低水平的表达。脑、肌肉、卵巢、肾脏、脾脏、鳃和心脏等组织中其表达量更低。而在脂肪和前肠中则没有检测到Lb-FabpmRNA表达。Lb-Fabp mRNA 最早在胚体形成期检测到有低水平表达,随后的发育阶段中表达量逐渐升高。鲤鱼Lb-Fabp基因的表达图式提示在肝脏和肠等器官开始发育后,它可能在脂肪代谢中具有重要作用。     

Sperm production and steroidogenesis in testes of the common carp, Cyprinus carpio L., at different stages of maturation

Testes from carp, Cyprinus carpio L., at five different maturational stages from immature through to spermiation and regression were incubated with or without addition of carp hypophysial homogenate (chh) for 8 or 20 h. Concentrations of steroids and spermatozoa were measured in the medium and the residual tissue examined histologically. There was an increase in the area of the germinal cysts containing spermatozoa, the percentage of the testis which they occupied and in the production of spermatozoa as the gonadosomatic index (GSI) increased, but this was unaffected either by incubation or by pretreatment with chh. The major steroid in plasma and in in vitro testicular cultures from all of the maturing fish captured in winter was 1 I-ketotestosterone. The production rate of this steroid in virro was unaffected by GSI, while plasma levels tended to increase with GSI. 17.20β-Dihydroxy-4-pregnen-3-one was detectable in significant amounts in only a few spermiating fish in summer, but was stimulated more in incubations with chh in maturing winter than in summer spermiating or post-spawning fish. 17,20a-Dihydroxy-4-pregnen-3-one was not detectable in incubations, but plasma concentrations tended to increase towards spermiation and were positively correlated with the size of the cyst. After spawning, fish had low plasma steroid levels and failed to respond in vitro to pituitary extract, indicating a testicular post-spawning refractoriness.     

Divergent selection for antibody production in common carp (Cyprinus carpio L.) using gynogenesis

A base population (n = 101) of carp, consisting of a single hybrid cross, was immunized with the hapten-carrier complex DNP-KLH. to perform a divergent selection for antibody response. Measurement of the DNP-specific antibody response at 12 and 21 days postimmunization, allowed the classification of a low number of individual carp as early/high (10%) or late/low (13%) responders. Three individuals defined as early/high and three defined as late/low responding, were gynogenetically reproduced to obtain corresponding homozygous progenies within one generation only. Upon immunization with DNP-KLH, the antibody response was found to be significantly higher in the early/high responder homozygous offspring. Although the homozygosity of the offspring apparently caused a (s)lower antibody response (compared with the base population), the differences between the high and low responder offspring do indicate a genetic influence on the antibody response. The realized heritability (h2) for antibody production was estimated at 0.37 ± 0.36. The present study provides the basis for a divergent selection of homozygous inbred carp lines with a genetically controlled difference in antibody response. These inbred lines will allow us to investigate relationship(s) between immune responsiveness and resistance to infectious diseases in fish.     

Electroencephalograms and cerebral blood flow in carp, Cyprinus carpio, subjected to acute hypoxia

Changes in electroencephalogams (EEG) and cerebral blood flow were examined in carp immobilized with a muscle relaxant during 60 min hypoxia (water Po ₂ of approximately 20 mmHg) and subsequent 30 min normoxia. The amplitude of EEG waves recorded from the telencephalon decreased gradually but slightly with the progression of hypoxia, whereas the telencephalic blood flow increased mainly due to an increased blood velocity. These findings suggested that cerebral activity during hypoxia was compensated to some degree by increased cerebral blood flow. However, carp showed large variations in the patterns of EEG responses and cerebral blood flow.     

鲤鱼精子超微结构的研究

鲤鱼精子由头部,中片和尾部组成,头部的细胞核卵形,染色质致密。核中有些小空隙,空中的电子致密物质存在。中片紧连在核的后端。中片由中心粒复合体和袖套组成。中心粒复合体位于核后植入窝中,袖套一侧肥厚,一侧狭窄,袖套中有线粒体和囊泡。囊泡有二类,一类含有电子致密物质;另一类无电子致密物质。近袖套内膜处的细胞质中还存在着与内膜平行的膜,精子尾部从袖套腔中伸出。尾部的轴丝与基体相接。尾部的近核端多有许多囊泡     

Humoral immune response of carp Cyprinus carpio to Myxobolus artus (Myxozoa: Myxobolidae) infection

The circulating antibody which reacted with sonicated spores of Myxobolus artus was detected in some naturally infected carp. However, some other fish had no detectable sign of infection, though they had the antibody. When carp were injected either with intact or sonicated spores, the antibody was not produced, while fish injected either with developing stages (presporogonic and sporoblast stages) of the parasite or sonicated spores with bovine serum albumin elicited the antibody production. The results of the injection experiments suggest that (1) developing stages have antigenicity to carp, and (2) spores have lost the antigenicity; sonicated spores are haptens, with which the antibody can react. In an indirect fluorescent antibody technique, sera positive for the antigen reacted with developing stages of the parasite, but not with the spore.
The mechanism of the host immune response against M. artus is discussed in relation to a previous observation that the parasite sometimes underwent abnormal development, in which host encapsulation was imperfect or even lacking, probably leading to degeneration of pseudocysts before the completion of spore formation. It is plausible that the antibody was produced when pseudocysts which showed abnormal growth ruptured during their developing stages, resulting in exposure of the young parasite to the host immune system.     

铜对鲫鱼脾细胞超微结构的影响

应用透射电镜的方法,研究了三种铜离子浓度对鲫鱼脾细胞超微结构的影响。结果表明：脾细胞超微结构变化明显,主要表现在线粒体和内质网,线粒体双层膜解体,形成空白区,嵴断裂,膨大和扭曲,内质网亦变化明显。     

镜鲤体长性状的QTL定位分析

以镜鲤良种后代为祖父母本所培育的杂交F2群体的68个个体为材料,利用553个分子标记(217个SSR和336个SNP标记)对其进行基因型检测,运用JoinMap4.0软件包对遗传连锁图谱进行构建。利用MapQTL5.0区间作图法(Interval mapping)进行QTL检测,通过置换实验(1 000次重复)确定连锁群显著性水平阈值。在对体长的区间定位中共检测到12个与体长性状相关的QTLs区间,分布在BL-1-1(SNP0137-SNP1481)、BL-4-1(SNP0092-HLJ797)、BL-5-1(SNP1268-HLJ423)、BL-7-1(HLJ870-SNP0702)、BL-12-1(SNP0922-HLJ639)、BL-16-1(HLJE351-SNP0674)、BL-25-1(SNP0394-SNP0862)、BL-35-1(HLJ668-SNP0832)、BL-43-1(SNP0389-SNP1425)、BL-47-1(HLJ057-HLJ1113)、BL-47-2(HLJ1439-HLJ1418)等11个连锁群上,解释表型变异范围是13.8%~64.9%,其中贡献率大于20%的主效QTLs有8个,是体长性状的主效QTLs区间。     

Identification of a new granulocyte type in the skin of carp Cyprinus carpio (L.)

The skin of carp Cyprinus carpio (L.) was investigated by electron microscopy. A new granulocyte cell type was identified, and is described as being similar to the eosinophilic granular cell (EGC) of salmonids.