剑尾鱼IgZ基因的克隆及疫苗免疫对其在组织中表达的影响

为研究剑尾鱼Ig Z基因序列及其在疫苗免疫后的表达规律,本实验根据已知的EST库设计引物,利用RT-PCR等方法,获得剑尾鱼Ig Z基因c DNA全长序列,并进行生物信息学分析和疫苗免疫后组织表达分析。结果显示,剑尾鱼Ig Z基因全长序列为5 420 bp,包含4个外显子和3个内含子;c DNA序列全长1 527 bp,包含一个1 299 bp的完整ORF框,该基因编码432个氨基酸,预测其编码蛋白的分子量大小为47.48 ku,并具有Ig Z的基本结构,与其他硬骨鱼类Ig Z氨基酸序列一致性为28%~54%。荧光定量PCR分析结果显示,Ig Z基因主要在剑尾鱼的头肾、脾脏和肠中分布,且疫苗免疫后11天内,Ig Z基因在头肾、脾脏和肠组织中均表现为先上升后下降的趋势。头肾中Ig Z基因的表达量在第4天时最高,为对照组的2.12倍;脾脏中Ig Z基因在第4天时呈现最高峰,为对照组的4.65倍;肠组织中Ig Z基因的表达量在12 h时有一个小高峰,第2天时最高,为对照组的11.41倍。本研究获得了剑尾鱼Ig Z基因c DNA全长序列,并对其表达规律进行了初步研究,发现Ig Z在肠道黏膜免疫中具有重要作用,这将为进一步验证其在黏膜免疫中的作用以及剑尾鱼作为疾病研究模式动物和疫苗免疫评价模型奠定基础。     

利用18个微卫星座位对剑尾鱼RR-B系遗传质量的分析(英文)

[Objective] The study aimed to further understand the genetic quality of Xiphophorus helleri RR-B strain and estimate the feasibility of microsatellite markers in genetic monitoring of aquatic laboratory animals.[Method] Eighteen microsatellite loci which had polymorphism in wild X. helleri were used for genetic quality analysis of RR-B strain (red eyes and red body) by PCR. [Result] All these 18 microsatellite loci displayed single allelic gene band in 30 individuals of X. helleri RR-B strain and the average homozygote rate reached 100%. [Conclusion] These loci could be used as markers in genetic monitoring of X. helleri RR-B strain, which had obtained fairly high genetic homozygosity by inbreeding for more than 20 generations. This study provided basic data for establishing the molecular genetic monitoring standard of aquatic laboratory animals.     

微卫星标记在RR-B系剑尾鱼遗传监测中的应用

采用49对微卫星引物对RR-B系剑尾鱼和红眼红体的非选育系剑尾鱼进行PCR扩增，有46个微卫星座位能获得稳定的扩增产物，7个微卫星座位能区分RR-B系与非选育群体剑尾鱼。7个微卫星座位在选育系剑尾鱼为单态纯合，而在非选育群体具有多态性，座位Msa014鉴定RRB系剑尾鱼排除概率最高，为98.75%，座位Msd003排除概率最低达到了87.50%，其余5个座位排除概率介于两者之间。为方便今后的遗传监测，对RR-B系剑尾鱼样品的7个微卫星座位进行了测序，确定了其大小和具体的微卫星结构。本实验建立了RR-B系剑尾鱼分子检测方法，为实验动物剑尾鱼的遗传监测奠定了基础。图4表3参23     

溶藻弧菌感染对剑尾鱼HSP70基因表达的影响

用溶藻弧菌Vibrio alginolyticus感染剑尾鱼Xiphophorus helleri,取感染后第0、2、5、8、11 d的活鱼及感染后第2 d濒死鱼,采用半定量RT-PCR方法分别检测HSP70基因在肝胰脏、脾脏、头肾和心脏组织中的表达。结果表明:正常条件下,HSP70在检测的剑尾鱼组织中均不表达;在用溶藻弧菌感染第8 d的剑尾鱼肝胰脏内,HSP70基因被诱导强烈表达,HSP70/β-actin的值为1.80±0.03;在感染第2、5 d的剑尾鱼头肾内,HSP70基因被诱导表达,第8 d HSP70基因强烈表达,感染濒死鱼头肾内也检测到HSP70基因的强烈表达,HSP70/β-actin的值分别为0.35±0.02、0.15±0.01、2.00±0.06和0.95±0.05;在剑尾鱼被感染第2、5、8 d,在脾脏内均检测到HSP70基因的表达,感染濒死鱼脾脏内也检测到HSP70基因的强烈表达,HSP70/β-actin的值分别为0.30±0.02、0.15±0.01、0.45±0.03和1.55±0.04;仅在感染濒死鱼的心脏中检测到HSP70基因的表达,HSP70/β-actin的值为0.30±0.02;到第11 d HSP70基因表达消失。     

迟缓爱德华菌(Edwardasiella tarda)对小鼠和剑尾鱼的免疫保护试验

为了评价迟缓爱德华菌 (Edwardasiella tarda)的免疫保护效果 ,用致病性 E.tarda JEL4的全菌灭活物 (FKC)和胞外产物 (ECP)分别 2次免疫小鼠 15 0只和 1次免疫剑尾鱼 30 0尾 ,7周后用同源菌株和异源菌株进行攻击。结果显示 ,除 ECP组对某一异源菌株为 4/ 5保护外 ,免疫小鼠 FKC组和 ECP组全部获得保护 (5 / 5 )。在免疫的剑尾鱼中 ,FKC和 ECP组对同源菌株保护率分别为 6 0 %和 10 0 % ;对异源菌株的攻击 ,均不能全部保护 ,但 ECP组可明显延缓剑尾鱼死亡时间     

剑尾鱼线粒体DNA D-环及侧翼tRNA↑（thr）和tRNA↑（pro）序列与结构分析

采用PCR方法从剑尾鱼(Xiphophorus helleri)线粒体基因组中扩增到1段约15．5kb的片段，测定了其中627个碱基的序列。经测序分析表明，该扩增片段包括486bp的D—环的部分序列以及D—环5端的tRNA^trh和tRNA^pro基因的完整序列。其中486bpD环序列包含3段保守的终止相关序列TAS、与TAS互补的序列以及类似其他鱼类线粒体CSB序列。tRNA^trh由线粒体DNA的H链编码，长度为72bp。tRNA^pro由线粒体DNA的L链编码，长度为69bp。并绘制了这2种tRNA的二级结构图。本研究所测定的基因序列已登录国际GenBank数据库，序号为AF489918。     

Filial Cannibalism in the Swordtail Xiphophorus Helleri (Poeciliidae)

To understand behaviour during filial cannibalism in swordtails (Xiphophorus helleri), the parturition behaviour of eight individually kept females was recorded on videotape. Three levels of female activity (low, medium and high) were quantified by measuring the duration of each. Four additional behavioural events (non-cannibalistic feeding, as well as the birth, attack and cannibalism of juveniles) were quantified by determining the frequency of each. Females gave birth most frequently at a low level of activity and at this level of activity they most often gave birth at the top of the tank. Of all the attacks by females on the young, 84.4% occurred in the light. Most attacks were recorded at the bottom of the tank and 84.0% of all cannibalism occurred at the bottom. Cannibalism was most successful at the bottom of the tank with a cannibalism:attack ratio of 1:2.2 and least successful in the middle where no cannibalism was recorded. In the light, females spent more time at the top of the tank, whereas in the dark they spent more time at the bottom. It was suggested that the rate of cannibalism might be reduced if parts of the tank were kept constantly dark and if females were restricted from reaching those areas in the tank where the frequency of cannibalism was highest, namely the top and the bottom of the tank. Experimental designs and working hypotheses for future studies are discussed.     

剑尾鱼热应激蛋白HSP70 cDNA片段的克隆与序列分析

为了进一步开展剑尾鱼Xiphophorus helleri--标准化实验动物在抗逆方面的相关研究,以及热应激蛋白(HSP70)在抗逆方面的作用,作者用RT-PCR方法首次克隆得到了剑尾鱼的HSP70的cDNA片段,该片段包含HSP70的特征性序列(GAT CTC GGT GGT GGC ACT TTT GAT).通过将克隆片段与已发表的其它鱼类HSP70的核苷酸序列和其编码的氨基酸序列进行同源性比较,发现剑尾鱼核苷酸序列同源性较高,与牙鲆Paralichthys olivaceus HSP70核苷酸序列同源性最高为89%,与川鲽Platichthys flesus、虹鳟Oncorhynchus mykiss和黄锡鲷Rhabdosargus sarba的同源性皆在85%以上;与国外学者发表的HSP70的规律类似,所克隆的片段与其它鱼类HSP70基因编码的氨基酸序列同源性较相应核苷酸序列同源性更高,与牙鲆和川鲽的HSP70氨基酸序列同源性最高达到99%,与斑马鱼Danio rerio、虹鳟和黄锡鲷的氨基酸序列同源性均高于97%.不同鱼的HSP70 cDNA核苷酸序列存在一定差异,但氨基酸序列并未受到影响,经对照,这种不同相当一部分存在于密码子的第3个碱基,因为密码子的简并性,编码氨基酸没有发生改变.     

Effect of probiotic on reproductive performance in female livebearing ornamental fish

A probiotic bacterial strain, Bacillus subtilis, isolated from the intestine of Cirrhinus mrigala (Hamilton), was incorporated in fish feed at four different concentrations (5 × 10⁸ cells g⁻², 5 × 10⁷ cells g⁻², 5 × 10⁶cells g⁻¹ and 5 × 10⁵ cells g⁻¹) and fed to four species of livebearing ornamental fish, Poecilia reticulata (Peters), Poecilia sphenops (Valenciennes), Xiphophorus helleri (Heckel) and Xiphophorus maculatus (Gunther) for one year duration to observe the effect of dietary probiotic supplementation on their reproductive performance. Sixty virgin females of each species were stocked separately in circular FRP tanks (350 L) and fed diets with varying levels of probiotic cells and control feed. Broodstock performance was evaluated based on gonadosomatic index (GSI), fecundity and fry production of female broodstock. The results showed that supplementation of feed with probiotics increased significantly (P<0.05) the GSI, fecundity and fry production of spawning females and length and weight of fry in all the four species of fish. The number of dead and deformed fry were also significantly lower (P<0.05) in fish fed with the probiotic feeds. The use of higher concentration of the probiont in diet did not always lead to significantly improved reproductive performance of the spawners. Collectively, this study showed that female livebearers benefit from inclusion of probiotics in diet during their reproductive stages.