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,...     

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。     

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.     

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。     

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。     

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。     

牙鲆胚胎及其初孵仔鱼的盐度耐受力

实验盐度设置为５，１０，１５，２０，２５，３０，３５，４０，４５，５０。当盐度为５～３５时，牙鲆（Ｐａｒａｌｉｃｈｔｈｙｓ ｏｌｉ－ｖａｃｅｕｓ）胚胎的孵化率都高于９５％；仔鱼体态正常，７２ｈ后的存活率高于９５％。刚孵化的仔鱼在低盐度下的体长较长，但７２ｈ后，没有明显差异（Ｐ〉０．０５）。自然条件（盐度为３２）孵化的仔鱼转移到不同盐度的溶液中，在盐度５～４５内，仔鱼的存活率高于５０％。实验初步表明     

牙鲆(Paralichthys olivaceus) Activin 基因两种β亚基的启动子克隆及生物信息学分析

以牙鲆为研究对象,利用染色体步移(Genome walking)获得了 Activin 两个β亚基基因的上游部分启动子序列,并对其进行了转录调控元件的生物信息学预测分析。获得了 Activin βA 和βB两个亚基的启动子部分序列,长度分别约为2.7 kb 和2.4 kb;在预测的 Activin βB 转录起始位点(+1位)的上游31 bp 处有1个典型的 TATA box,而在 Activin βA 中未发现 TATA box 的存在。两个基因的启动子上发现了多个转录因子 Sp1、Oct-1、C/EBP、CREB、GATA-1、HNF-3、HNF-1、USF 等结合位点,还发现了与内分泌相关的 Pit-1、ER、PR、GR、RAR、RXR 等结合位点,但肌性转录因子 MyoD、myogenin 和雄性的性别决定基因 SRY 结合位点仅在 Activin βA 启动子中发现。生物信息学分析显示,牙鲆 Activin βA 和βB 表达受到多种潜在因子的调控,二者在调控上有所差异。     

变态前牙鲆cDNA文库中Ⅰ型微卫星的多态性分析

牙鲆(Paralichthys olivaceus)不仅是研究鱼类变态的模式生物,也是我国的重要水产养殖对象。从变态前牙鲆cDNA文库中的42个单一序列表达标签(ESTs)中筛选到46个微卫星分子标记,从中选择21个微卫星并在一个养殖牙鲆群体共30个样本中测试,结果表明其中有20个微卫星标记具有多态性,其等位基因数为2到6个,观察杂合度从0.06到1.00,期望杂合度从0.059 7到0.825 2变化不等,平均多态信息含量是0.457 5,可用于牙鲆连锁图的构建和分子标记辅助选育。其中,有3个微卫星位点的三核苷酸重复序列在基因编码区内,可能和基因功能有关。     

Can fermented soybean meal and squid by‐product blend be used as fishmeal replacements for Japanese flounder (Paralichthys olivaceus)?

An experiment was carried out to evaluate fermented soybean meal and squid by‐product blend (1:1) (FP) as replacement of fishmeal (FM) for Japanese flounder, Paralichthys olivaceus. Five isocaloric (19 kJ g^?1) diets were prepared by replacing 0 (FP0), 12 (FP12), 24 (FP24), 36 (FP36) and 48% (FP48) FM protein with FP. Triplicate groups of juveniles (mean weight of 3.9 g) were delivered the test diets for 8 weeks in a flow‐through sea water system. The results showed that there were no significant differences (P > 0.05) among the growth rates of fish fed FP0, FP12, FP24 and FP36 diets. Growth and nutrient utilization parameters were significantly reduced in fish fed FP48 diet. Although, whole body proximate composition of fish were not significantly affected by the dietary treatments compared to the control; methionine and phenylalanine contents were significantly decreased in FP48 group. Protein retention was also significantly decreased in the similar group of fish. Dietary treatments did not alter most of the plasma metabolites, while some of the health parameters were improved in the replacement groups. Results suggested that FP is a potential candidate for alternative protein ingredient in aquafeed and can replace 36% FM protein in the diet of Japanese flounder.