稀有鱼句鲫β肌动蛋白启动子的克隆及其驱动活性的初步检测

稀有鮈鲫是一种新型的模式实验鱼,具有利用转基因技术培育开发成为观赏性鱼类的潜在可能。本实验采用PCR方法,从稀有鮈鲫基因组DNA中分离得到大小为1584bp的β肌动蛋白(β-actin)基因片段。序列分析表明,该片段包括长为1507bp的启动调控区和77bp的部分转录区序列。启动调控区包括一段长为109bpβ-actin基因上游调控序列,不翻译的外显子1和内含子1。上游调控序列中含有TATAbox,CAATbox和CArGbox等与转录活性密切相关的作用元件,并且在稀有鮈鲫β-actin基因第一个内含子中也含有1个CArG调控元件。将该启动子片段克隆到绿色荧光表达载体(pAcGFP1-1)上,显微注射入稀有鮈鲫的受精卵中,通过荧光显微镜能观察到绿色荧光蛋白的表达。本实验成功分离了具有驱动活性的稀有鮈鲫β-actin基因启动子。     

稀有鮈鲫β-肌动蛋白启动子的克隆及其驱动活性的初步检测

稀有鮈鲫是一种新型的模式实验鱼,具有利用转基因技术培育开发成为观赏性鱼类的潜在可能。本实验采用PCR方法,从稀有鮈鲫基因组DNA中分离得到大小为1584bp的β肌动蛋白(β-actin)基因片段。序列分析表明,该片段包括长为1507bp的启动调控区和77bp的部分转录区序列。启动调控区包括一段长为109bpβ-actin基因上游调控序列,不翻译的外显子1和内含子1。上游调控序列中含有TATAbox,CAATbox和CArGbox等与转录活性密切相关的作用元件,并且在稀有鮈鲫β-actin基因第一个内含子中也含有1个CArG调控元件。将该启动子片段克隆到绿色荧光表达载体(pAcGFP1-1)上,显微注射入稀有鮈鲫的受精卵中,通过荧光显微镜能观察到绿色荧光蛋白的表达。本实验成功分离了具有驱动活性的稀有鮈鲫β-actin基因启动子。     

唐鱼β-肌动蛋白基因启动子的分离及其驱动活性的检测

利用PCR技术克隆唐鱼（Tanichthys albonubes）β-actin基因。所克隆的β-aetin基因片段为1464bp，包含长为1374bp的启动调控区和90bp的部分开放阅读框。启动调控区包括105bp的β-actin基因上游调控序列、第一个外显子和第一个内含子。上游调控序列中含有对转录起重要作用的CAAT Box、TATA Box、CArG Box等元件。将唐鱼β-actin启动调控区克隆到红色荧光表达载体pDsRed2-1上，并显微注射到唐鱼受精卵中，荧光显微镜观察红色荧光蛋白（RFP）的表达。结果表明，RFP在转基因唐鱼中的表达阳性率较高，最高可达51．8％，且RFP的表达水平较高。PCR检测转基因唐鱼的部分器官组织，在被检组织器官中均能检测到外源RFP基因；而RT-PCR以及Southern blot验证显示RFP mRNA的表达有所不同；Southern blot检测肌肉组织基因组DNA，可见比阳性载体大的杂交条带。说明所检测的组织中已发生外源基因RFP的整合，但有些组织存在表达水平较低或者不表达的现象。本实验分离到的β-actin基因启动子序列具有有效的驱动功能，可启动外源基因在唐鱼体内的高效表达，从而为下一步进行功能基因的转化研究奠定基础。     

鲮β-肌动蛋白基因的分子克隆及其作为分子内标的可靠性分析

β-actin是actin家族的一员，在维持细胞结构、细胞内运动和细胞分裂等细胞生理活动方面发挥着重要的作用。克隆的鲮（Cirrhinus molitorella）β-aetin基因的cDNA全长1 804bp，开放阅读框长1 128bp，编码375个氨基酸，5’和3’末端非翻译区域（UTR）分别为94bp和582bp。RT-PCR结果表明，β-actin在未经LPS刺激的和经LPS刺激的鲮心脏、肾脏、肌肉、肝脏、脾脏、肠、脑和皮肤等8种组织中广谱表达，表达水平没有明显差异，可以作为基因表达调控研究的分子内标。鲮与其他鱼类β-actin基因编码区核苷酸序列同源性在85.9％～99.6％。分子系统进化树分析表明，可将鱼类分成鲤形目，鲈形目，鲑形目和合鳃目4大支，与传统分类一致，β-actin基因核苷酸序列是研究分子进化的一个很好的分子标记。     

斑节对虾亲环素A （cyclophilinA） 基因的克隆及启动子序列分析

通过同源克隆和RACE技术获得斑节对虾(Penaeus monodon)亲环素A(cyclophilin A,CYPA)的cDNA序列.根据已克隆的cDNA序列设计引物,利用染色体步移技术(Cenomic DNA walking)从斑节对虾卵巢组织中克隆了CYPA基因的启动子和基因组DNA序列.测序结果表明,斑节对虾亲环素A(PmCYPA)cDNA全长834 bp,其中开放阅读框(open reading frame,ORF)为495 bp,可编码164个氨基酸;5'非编码区为31 bp,3'非编码区为308 bp.从斑节对虾基因组文库中扩增的CYPA基因组DNA全长3 181 bp,其中包括启动子区域1 173 bp,1个内含子426 bp,2个外显子序列:分别为101 bp和399bp.在5'UTR上游区域有明显的启动子序列,包含一个GC盒和2个CAAT盒,同时还包含AP1、CRE等调控元件,符合真核生物典型的启动子特征.分析基因的结构表明所克隆的PmCYPA符合PPlase家族成员特征,属于此基因家族成员.     

泥鳅β-actin启动子介导的革胡子鲶GH重组基因的构建及启动活性研究

为获得快速生长的泥鳅(Misgurnus anguillicaudatus),研究了泥鳅β-actin基因启动子介导的革胡子鲶(Clarias gariepinus)生长激素(growth hormone,GH)重组基因。采用PCR和RT-PCR技术克隆泥鳅β-actin基因近端启动子、3′-UTR及革胡子鲶GH基因编码区,构建一个长为2 418bp的GH重组基因DPRK。首先,构建了以增强型绿色荧光蛋白(EGFP)基因作为报告基因的重组表达载体p AF(pβ-actin promoter-EGFP),然后转染DF1真核细胞,同时经酶切线性化后显微注射到斑马鱼(Danio rerio)和泥鳅的受精卵中,以评价泥鳅β-actin启动子的启动活性。其次,将重组基因DPRK显微注射到泥鳅受精卵中。结果显示:泥鳅β-actin启动子能在DF1细胞、斑马鱼和泥鳅的受精卵中启动绿色荧光蛋白的表达;泥鳅的荧光表达率(78.44%)显著高于斑马鱼(29.62%);泥鳅受精卵显微注射DPRK 20d后,在m RNA水平检测到GH基因的表达。这表明泥鳅β-actin基因近端启动子具有显著的启动活性,且重组基因DPRK能在泥鳅体内表达,为下一步泥鳅的基因工程育种奠定了理论基础。     

虹鳟免疫诱导型基因Cathelicidin2启动子功能分析

本研究通过实时荧光定量PCR实验对虹鳟(Oncorhynchus mykiss)Cathelicidin2(Cath2)基因的转录模式进行了分析。结果显示,该基因在鳃、头肾等与机体免疫防御功能密切相关的组织内转录,在细菌和病毒感染后,转录水平均显著升高。对基因上游调控序列进行启动子和转录因子结合位点预测,发现该启动子具有真核生物典型的TATA盒和CAAT盒结构,基因上游直至第一内含子区域内,密集存在多个免疫相关转录因子结合位点,其中,2个核因子κB(Nuclear factor kappa B,NFκB)预测结合位点均位于核心启动子正链区域。在草鱼(Ctenopharyngodon idellus)肾组织细胞系内,绿色荧光蛋白和萤火虫荧光素酶基因都能够在该启动子驱动下表达,表明其具有启动子活性,且启动子活性在受到免疫诱导后增强,包括细菌脂多糖(Lipopolysaccharides,LPS)模拟的细菌感染和聚肌胞苷酸(Polyinosinic polycytidylic acid,Poly I:C)模拟的病毒感染。双荧光素酶报告基因检测显示,启动子活性在与NFκB转录因子表达时,增强至4.39倍,证明Cath2基因受NFκB通路调控。研究表明,虹鳟Cath2基因能够在多种免疫刺激诱导下表达,其启动子可以应用为免疫诱导型的基因工程元件,驱动外源免疫基因在鱼体内适时表达,抵御外界病原感染,同时,避免非必要条件下的过度表达形成生长负担。     

尼罗罗非鱼β-actin基因启动子的分离及其在真核细胞中的活性验证

采用高保真PCR方法从尼罗罗非鱼(Oreochromis niloticus)基因组DNA中分离出β-actin基因启动子序列,将β-actin基因启动子插入pN1-EGFP构建成真核细胞表达载体pEGFP-β-actin,并通过脂质体转染法将重组载体导入人胚肾上皮细胞HEK 293T,荧光显微镜下观察外源基因EGFP...     

“全鱼”Mx基因重组构建与转导分析

通过分子重组将鲫Mx1基因的编码序列克隆到鲤β肌动蛋白基因启动子下游,构建成能在鱼体内表达的转“全鱼”Mx基因的重组分子.采用显微注射方法,将含有鲤β肌动蛋白基因启动子和鲫Mxl基因序列的重组片段转入草鱼受精卵中,经人工孵化和养殖获得转全鱼Mx基因草鱼.经PCR和Southern杂交检测分析,证实重组分子成功整合到2月龄转基因草鱼基因组中,为草鱼抗病育种研究奠定了基础.     

大口黑鲈MyoD基因结构和单核苷酸多态性位点的筛选

本研究采用PCR技术和基因组步移技术从大口黑鲈基因组DNA中扩增得到MyoD基因及其5’调控区序列。该基因序列全长3797bp，其中5’调控区长1077bp，MyoD基因转录区由3个外显子（分别为591bp、81bp和78bp）和2个内含子（分别为1077bp和486bp）组成。5’调控区含有与肌肉特异性基因转录密切相关的转录调控元件E box、肌细胞增强因子2（MEF2）、肌肉特异性金属硫蛋白结合位点（MTBF）及一些转录反应调控元件（TATA box 、OCAAT box、OCT1、PRE、AP4、Pit1）。运用PCR-SSCP技术和直接测序法进行大口黑鲈MyoD基因SNP位点筛选，结果表明MyoD基因序列中存在7个突变点，均位于内含子上。养殖群体中这7个突变点分析结果显示突变比例范围在0.042～0.353之间。本研究结果为SNPs位点与大口黑鲈生长性能关联分析奠定了基础。     

Characterization of protease inhibitors of seminal plasma of cyprinids

Anti-proteinase activity was demonstrated in the seminal plasma of cyprinid fish species (bream, chub, ide, dace, asp, goldfish, roach, common carp) using electrophoretic techniques combined with a detection method based on inhibition of bovine trypsin. We found species-specific protease inhibitors in the seminal plasma of cyprinids. At least three bands of protease inhibitors with different migration rates could be identified by native PAGE. Higher variability was characterized for bands with slower migration rates. Visualization of inhibitors after SDS-PAGE under non-reducing conditions allowed estimation of their molecular weights. Apparent molecular weights were within the range of 51–59 and 47–54 kDa for the bands with slower and moderate migration rates, respectively. The molecular weight of fast migration bands for roach and common carp were estimated to 23 and 30 kDa, respectively. Inhibitors of common carp seminal plasma differed in their affinity toward serine proteases. Three inhibitors in common carp seminal plasma could be visualized using cod and bovine trypsin, but only two inhibitors (of high molecular weight) were recognized with chymotrypsin. There were differences in anti-proteinase activity and seminal plasma protein concentration in relation to the origin of common carp seminal plasma (breeding lines) and time of milt collection (spawning vs. post-spawning season).     

Estimation of variation of virulence of Renibacterium salmoninarum by survival analysis of experimental infection of salmonid fish

The variation of virulence of Renibacterium salmoninarum , the causative agent of bacterial kidney disease (BKD) in salmonid fish, was studied by infecting rainbow trout, Oncorhynchus mykiss (Walbaum), with two isolates (strains 325 and 932) from diseased Atlantic salmon, Salmo salar L., and one isolate (strain 4366) from an apparently healthy Atlantic salmon. Coho salmon, Oncorhynchus kisutch (Walbaum), were injected with the strain 932 to estimate difference in fish species resistance. Fish were removed by random sampling for other study purposes, a study design possible with analysis of lifetime distributions incorporating both sampling-, death- and survival-times. At the end of the experiment, the rainbow trout infected with strains 325, 932 and 4366 had a survival probability of 33%, 51% and 72%, respectively. The coho salmon infected with strain 932 had a 26% survival probability. The strain differences were significant according to the log-rank test, and the risk ratio between the strains ranged from 1·8 to 5·4. The strain from the apparently healthy fish was least virulent. The survival of the fish species was different over time. Rainbow trout were more likely to die early in the time course, but high numbers of coho died later, resulting in an overall risk of mortality of 1·4 in favour of rainbow trout. Differences in virulence may reflect changed selective pressure on R. salmoninarum when introduced from feral stocks into the environment of fish farms.     

母猪胎衣不下的病因与防治要点

猪的胎盘属于弥散型胎盘,这种胎盘的结构特点和饲养管理的不当,常常导致母猪胎衣不下发生,给生猪的生产繁殖带来极大损失。本文针对母猪胎衣不下发生病因、综合防治进行详细阐述,旨在对预防和治疗胎衣不下能有所帮助。     

水硬度对七彩神仙鱼幼鱼发育的影响

采用不同硬度的水对七彩神仙鱼幼鱼进行饲养。6周龄幼鱼在硬度为7.94°dH±0.30°dH时饲养84d后,比在硬度为14.71°dH±0.23°dH水中的幼鱼个体大,生长速度快。表明较高硬度的水有利于七彩神仙鱼幼鱼的生长发育。     

虎斑乌贼受精卵卵黄营养成分分析

本实验对虎斑乌贼受精卵卵黄的营养成分进行分析,旨在探讨其幼体的营养需求量,为其幼体配合饲料研制提供参考数据。随机选取大约800个虎斑乌贼受精卵的卵黄,采用国家标准方法测定其水分、灰分、粗蛋白质、粗脂肪、氨基酸、脂肪酸和矿物元素含量。结果表明:1)虎斑乌贼受精卵卵黄中粗蛋白质含量为76.33%(干重基础);总氨基酸(TAA)和必需氨基酸(EAA)含量分别为71.22%和32.38%(干重基础),EAA/TAA为45.46%,氨基酸中以谷氨酸(Glu)含量最高(9.97%),必需氨基酸中亮氨酸(Leu)含量最高(7.58%)。2)其粗脂肪含量12.71%(干重基础);共检出17种脂肪酸,包括8种饱和脂肪酸(SFA)、5种单不饱和脂肪酸(MUFA)和4种多不饱和脂肪酸(PUFA),SFA、MUFA和PUFA分别占脂肪酸总量的43.47%、7.54%和49.25%,其中以DHA含量最高,达32.80%,EPA含量为7.70%,DHA/EPA为4.26。3)检测出Na、K、Ca、Mg、Sr、Mn、Fe、Cu、Zn、Al和As 矿物元素,微量元素中富含Zn、Al和Fe,含量分别为 0.77、0.71和0.43 mg/kg(鲜重基础)。由此可见,卵黄具有高蛋白、低脂肪,富含n-3PUFA的特点;虎斑乌贼幼体饲料中蛋白质需求量参考值为76.33%;氨基酸需求量参考值,如赖氨酸(Lys)为5.49%,蛋氨酸(Met)为2.63%;脂肪的需求量参考值为12.71%,DHA为4.17%,EPA为0.98%;微量元素需求量参考值,如Zn为2.77 mg/kg,Cu为0.19 mg/kg(干重基础)。     

头足类耳石微化学研究进展

耳石是位于平衡囊内起平衡作用的一对钙化组织,它是头足类的加速度感应器,记录其生命周期内的生物和生态信息。随着鱼类耳石微化学研究及应用的日趋成熟与完善,头足类耳石的相应研究也逐渐兴起。目前头足类耳石微化学的研究内容主要包括无机和有机大分子、微量元素、同位素、微化学标记等方面,其中微量元素是应用研究的重点,在头足类种群识别、生活史分析及栖息环境重建等方面发挥了重要作用。分析认为,头足类微量元素在与栖息环境尤其水温关系的研究中取得了很好的结果,被认为是测定头足类生活水温的温度计。然而,涉及种群识别、生活史分析以及与盐度和食物关系的研究还不够充分,且多集中于Sr/Ca的研究。因此,建议在今后的研究中要综合多种研究方法按时间和空间序列从日轮水平分析多种微量元素的含量与变化。     

不同品系盐生杜氏藻培养技术的研究

4种不同品系盐生杜氏藻培养结果表明：常温下,A33是最适宜培养的藻种,经4天培养,藻细胞密度可达到114×104cell／ml,细胞生长率达到0．369;其次是A23、A140、A5藻种,细胞生长率分别为0．317、0．314、0．234。控温条件下,4种品系杜氏藻细胞生长速度明显增高,是常温条件下的1．2倍。     

Histopathology of respiratory organs of certain air-breathing fishes of India

Air-breathing fishes have evolved bimodal respiratory mechanisms for exploitation of water (through gills and highly vascularized skin) as well as atmospheric air (through aerial respiratory organs, ABO). Mucous cells in these respiratory organs of variously stressed fishes exhibit periodic fluctuations in their density and staining properties. The main types of damage in the gills include congestion of blood capillaries (BLCs), periodic lifting and sloughing of respiratory epithelia of the secondary lamellae causing haemorrhage, extensive fusion of secondary lamellae and hyperplasia of the respiratory epithelia due to uncontrolled regeneration leading to asphyxiation, altered excretion, and death of the fish. Haemolysis has also been observed following lead exposure. The damage in the ABO of Heteropneustes fossilis includes sloughing of the epithelial cells, leading to haemorrhage causing decreased red blood corpuscles density and degeneration of the secondary gill lamellae with reduced respiratory area. Subsequent hyperplasia of the respiratory epithelia and fusion of gill lamellae increase the respiratory barrier distance. The BLCs often bulge out and protrude into the lumen, bringing blood nearer to air. The ladder-like pillar cell (PLC)-BLC components of the gill lamellae frequently collapse. Damage to the ABO of Channa striata is less severe. Often haemorrhaging due to bursting of extensively stretched BLCs causes aerial respiratory failure. Chloride cells of the ABOs also show hyperplasia. While the highly mucogenic epidermis of C. striata shows less damage, the epidermis of Clarias batrachus and H. fossilis shows severe wear and tear, sloughing, and haemorrhage. Side-by-side regeneration continues, causing altered histomorphology of the epidermis. The different gland cells also show periodic fluctuations in their density and staining. The dermis also shows severe damage with loosening of their connective tissue fibres. These fibres give stronger reactions for sulfated mucin that not only retain additional water molecules for continuance of skin breathing, but also bind the toxic ambient pollutants.     

鱼类热忍耐温度研究进展

The paper is a review of the studies on the temperature of the thermal tolerance in fishes. It was described from four aspect: (1) The key parameters of temperature of thermal tolerance in fishes; (2) The influence factors of temperature of thermal tolerance in fishes; (3) The research methods of thermal effect of fishes; (4) The evaluation of effects of thermal pollution on fishes, especially, with the coastal power station will be‘built in China recent years, the effects of thermal discharge water on the marine fisheries resource wered iscussed.     

台风对江苏近海海表温度的影响分析与探讨

利用中国台风网"CMA-STI热带气旋最佳路径资料数据"和多卫星遥感观测资料,本文对2011年09号梅花台风、2011年06号马鞍台风和2012年10号达维台风在发展过程对中国江苏近海海域海温的影响进行了分析与探讨,并辅助以沿岸的海温实测资料进行对比验证,借此提高对近海海域上层海洋物理环境对台风响应的认识。分析结果表明,台风对近海SST的影响程度与台风自身强度和台风的移动路径密切相关,近海SST降低区域一般位于台风路径的右侧,最大SST降低一般滞后台风中心两天或一天。当台风在远海活动并靠近江苏近海时,江苏近海海域SST略有上升;当台风进入江苏近海时,近海海域SST大幅降低;登陆台风在登陆后近海才出现最大降温。