牙鲆let-7基因的克隆与表达及其靶基因预测

let-7是时序发育过程中的重要调控因子,在促进幼体向成体转变过程中起着极其重要的作用.为探讨let-7对牙鲆(Paralichthys olivaceus)从幼体到成体转变的变态发育过程的影响,本实验对let-7进行了克隆与表达研究,并预测了其可能的靶基因.利用普通PCR技术从牙鲆中成功鉴定出let-7及其前体序列,...     

牙鲆微卫星标记的开发及多态性分析

将牙鲆基因组DNA经限制性内切酶RsaⅠ和BstUI双酶切后采用FIASCO法构建酶切片段基因组文库。共挑取269个克隆,177个为阳性克隆,阳性克隆率为65.79%。经测序后获得191条微卫星序列,其中完美型占74.35%;非完美型占14.66%;混合型占10.99%。用引物设计软件Primer Premier 5.0设计引物153对,挑选其中的50对合成并在32个野生牙鲆个体中进行扩增,共31个位点具有多态性,统计结果后使用POPGENE软件进行分析,平均等位基因个数为3.939 4,平均有效等位基因数为3.052 2,平均观测杂合度为0.650 1,平均期望杂合度为0.586 6,各引物的Hardy-Weinberg平衡指数在0.012 587~0.984 917变动。这些筛选出的多态性微卫星标记可应用于进一步的牙鲆遗传多样性分析、家系分析及遗传图谱的构建等工作中。     

牙鲆群体生化遗传学研究——Ⅰ.组织特异性研究

采用聚丙烯酰胺凝胶电泳技术,研究了AAT、EST、SOD、MDH、LDH和ME等位酶在牙鲆[Paralichthys olivaceus(T.et S.)]心脏、肌肉、鳃、眼睛、肾脏、肝脏、肠、脾、胃组织器官中表达,并分析了各种酶的等位酶位点表达以及酶谱表型。研究结果表明EST、SOD、MDH和ME 4种等位酶在不同组织中表达相同,AAT、LDH 2种等位酶在不同组织中表达差异,可能是受不同基因位点或同一基因位点不同等位基因控制的结果,与各组织完成特有生化代谢活动有关。     

高温胁迫对褐牙鲆生长及肝脏抗氧化酶活性的影响

为了研究高温对褐牙鲆幼鱼生长及抗氧化酶活性的影响,对其不同温度处理(25、28、30、31、32℃)下的存活、生长、摄食及超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活力进行比较。结果显示25℃组、28℃组及30℃组的鱼没有死亡,31℃组的成活率为(95%±1.18%),32℃组的成活率仅为(20.83%±2.20%)。各温度处理组的特定生长率(SGR)、摄食率(FR)和饲料转化率(FCE)和水温的平方呈显著的负相关(SGR:R2=0.93;FR:R2=0.90;FCE:R2=0.86;P<0.01),即25～32℃时,特定生长率、摄食率和饲料转化率随温度升高而减小。各处理组的肝脏组织中的SOD及CAT活力随胁迫温度升高而降低。本研究表明32℃是褐牙鲆长期生存的临界上限温度,高温能够显著影响其生长和抗氧化酶的活性。     

3种鲆鲽鱼精子和卵子表面形态结构的比较研究

使用扫描电镜对牙鲆、大菱鲆和条斑星鲽的精子和卵子表面形态结构进行比较研究。观察结果表明,3种精子形态结构在核前凹陷位置、精子头部形状、中段线粒体数及鞭毛长度4个方面存在明显差异;3种卵子表面形态结构中,牙鲆和大菱鲆比较相似,而条斑星鲽成熟卵子壳膜具有4点特异性:外表面粗糙、有点状和斑块状突起、网纹较深;壳膜上微小孔孔径差距大;卵膜厚度最厚为(3.87±0.24)μm,约为牙鲆和大菱鲆卵膜厚度的2倍;受精孔的管道螺旋层为4~6层,约为前两者的1/2。条斑星鲽与牙鲆和大菱鲆的精卵表面形态结构差异明显。     

Efficacy of Sarafloxacin Hydrochloride in Treating Naturally-Induced Edwardsiella ictaluri Infections in Channel Catfish,Ictalurus punctatus

Sarafin (sarafloxacin hydrochloride), a new antibacterial, was evaluated in the field on a naturally induced infection of Edwardsiella ictaluri in channel catfish, -Ictalurus punctatus. Healthy channel catfish (mean weight = 50 g) were stocked into nine cages at 200 fish per cage in a pond with an undergoing E. ictaluri infection. Seven days after stocking, dead fish were observed in the cages with clinical signs of enteric septicemia of channel catfish (ESC). After E. ictaluri was confirmed through isolative biochemical tests, medicated feed was applied for five consecutive days. During this period, fish in three control cages received a commercial 32% protein floating feed, three other cages of fish served as positive controls and were fed Romet, and three cages received the test feed with Sarafin. Both medicated feeds reduced the increase in cumulative percent mortality. In the control cages, cumulative percent mortalities continued to increase throughout the study period. Average daily mortality rates were significantly lower following both treatments of medicated feed, and treatments receiving Sarafin showed the greatest reduction in average daily mortality rates. Average daily mortality rates in the control did not change after the medicated feeding period. Toward the end of the study, temperatures reached 30°C, above the active range of ESC infections, and all mortalities ceased.     

牙鲆仔鱼早期阶段的摄食能力

本文主要在实际观察值的基础上，利用发育反应方程Ｃ＝ＫＷ＾ｙ与功能反应Ｈｏｌｌｉｎｇ、Ｉｖｌｅｖ方程对２～１０日龄的牙鲆仔鱼的摄食能力进行了计算与预测。其结果表明：牙鲆仔鱼在２～６日龄、轮虫饵料密度不超过３０００ｉｎｄ．Ｌ＾－１的水体中，摄食强度不超过１．７μｇ．ｈ＾－１；随着日龄的增加，摄食强度迅速增强，在４、６、１０日龄、饵料密度为３０００ｉｎｄ．Ｌ＾－１的水体中，摄食强度分别是１．３５、１．６     

牙鲆碱性磷酸酶基因调控序列的功能分析及甲状腺激素对其调节作用

为分析牙鲆碱性磷酸酶基因5'调控区和第一内含子调控序列的功能,本研究运用DNA重组技术将PCR扩增得到的牙鲆碱性磷酸酶基因的5′调控区序列、第一内含子序列及5′调控区和第一内含子串联序列分别插入启动子缺失的增强型绿色荧光蛋白表达载体pAcGFP1-1中,得到pAcGFP1-5′ALP、pAcGFP1-Intron1和pAcGFP1-AI报告基因表达载体。通过脂质体介导重组质粒转染中国仓鼠卵巢细胞(CHO)并检测荧光信号。转染后细胞状态良好,24 h后发现,在倒置荧光显微镜下均可看到不同强度的绿色荧光信号,且荧光信号随时间的延长而增强,其中转染pAcGFP1-AI后的荧光信号强度最强。以上结果表明,牙鲆碱性磷酸酶基因5′调控区和第一个内含子序列均具有启动子活性,且第一内含子与5′调控区存在协同效应,两者协同能增强基因的表达。为了解甲状腺激素T3对调控序列启动子活性的调节作用,用不同浓度的T3处理转染的CHO细胞,24 h后发现加入50、75和100 nM的T3都增强了绿色荧光蛋白的表达,而且信号强度随T3浓度的增加而增强。这表明甲状腺激素T3对牙鲆碱性磷酸酶基因调控序列的启动子活性有一定的增强作用。本研究为深入阐明牙鲆碱性磷酸酶基因转录表达的分子机制提供了实验依据。     

Susceptibility by amberjack (Seriola dumerili), yellowtail (S. quinqueradiata) and Japanese flounder (Paralichthys olivaceus) to Neobenedenia girellae (Monogenea) infection and their acquired protection

This study investigated: 1) susceptibility differences to infection by Neobenedenia girellae (Capsalidae) between amberjack Seriola dumerili (Carangidae), yellowtail S. quinqueradiata and Japanese flounder Paralichthys olivaceus (Paralichthyidae); 2) growth and egg production of N. girellae on each fish species; 3) acquired protection of each fish species against this parasite. The number of N. girellae on S. dumerili was significantly higher than on S. quinqueradiata and P. olivaceus when these fishes were exposed to oncomiracidia in the same aquarium. Neobenedenia girellae growth on S. dumerili was fastest and, thus the number of eggs laid by parasites on S. dumerili was greater than on the other two species. Seriola dumerili and P. olivaceus, which were previously infected with N. girellae and treated by freshwater bath, acquired partial protection against re-infection by N. girellae. The relative re-infection of three S. dumerili individuals out of eleven individuals was markedly low compared with the initial infection, and the relative initial infection and re-infection on two P. olivaceus out of eleven individuals was markedly low. The results of this study could be useful to control N. girellae infections when cultivating S. dumerili, S. quinqueradiata and P. olivaceus.