大口黑鲈脂代谢相关基因NPY、UCP2、LPL、HL克隆与分子进化分析

采用RT-PCR和RACE方法克隆了大口黑鲈NPY基因cDNA全序列及UCP2、LPL、HL基因cDNA核心片段。序列分析结果表明,大口黑鲈NPY基因cDNA全序列长664 bp,其中5′端非翻译区(5′-UTR)长53 bp,3′端非翻译区(3′-UTR)长311 bp,开放阅读框(ORF)长300 bp,编码99个氨基酸,即前体NPY。大口黑鲈前体NPY包括三个部分,28个氨基酸组成的信号肽、36氨基酸组成的成熟NPY以及32个氨基酸组成的由Gly-Lys-Arg指示的NPY C端肽（CPON）。大口黑鲈UCP2、LPL、HL基因cDNA核心片段长度分别为737 bp、509 bp和666 bp,各自编码245个氨基酸、169个氨基酸和222个氨基酸。将4个基因的氨基酸序列分别与其他物种的氨基酸序列进行同源性比较,并通过MEGA3构建系统树,对这4个脂代谢相关基因的分子进化特征进行了探讨。     

红罗非鱼MyoD基因克隆及序列分析

MyoD基因是MRFs家族中的一员,能调控肌肉细胞活性和增殖,在肌肉形成过程中起正调控作用。采用同源基因克隆和RACE方法获得红罗非鱼Myo D基因的c DNA序列。开放阅读框长903 bp,编码300个氨基酸,其中第1-114个氨基酸为碱性结构(Basic domain),第109-165个氨基酸为螺旋-环-螺旋结构(Helix-Loop-Helix domain),第199-213个氨基酸为周期蛋白依赖性蛋白激酶4(CDK4)结合区域,第233-249为Helix III结构,无信号肽序列。Myo D蛋白的二级结构为螺旋-环-螺旋二聚体。基于Myo D构建的鱼类系统进化树显示红罗非鱼,尼罗罗非鱼和奥利亚罗非鱼聚为一支。     

关岭牛MyoD基因CDS区的克隆、序列分析及其真核表达载体构建

[目的]通过关岭牛MyoD基因CDS区的克隆、序列分析及pcDNA3.1(+)-MyoD真核表达载体的构建,为后续研究MyoD基因的调控机制奠定基础。[方法]通过设计特异性引物克隆关岭牛MyoD基因的CDS区,并用DNAStar、Ex PASy等软件对该基因CDS区进行序列分析;同时利用双酶切的方法构建真核表达载体pcDNA3.1(+)-MyoD。[结果]关岭牛MyoD基因的CDS区全长957bp,编码318个氨基酸;其编码的蛋白属于亲水性蛋白,蛋白二级结构主要以α-螺旋及无规则卷曲为主,含有b HLH结构域,无明显的跨膜区域。同源性分析显示,关岭牛MyoD基因的CDS区序列与海福特牛和山羊的同源性最高,核苷酸同源性为98.96%和96.9%,氨基酸同源性为100%和89.3%。MyoD蛋白的氨基酸肽链长度由低等动物到高等动物有逐渐加长的趋势。[结论]成功克隆了关岭牛MyoD基因的CDS区,并构建了其真核表达载体pcDNA3.1(+)-MyoD,为进一步研究MyoD基因在成肌分化过程中的作用及其调控机制奠定基础。     

大口黑鲈北方亚种和佛罗里达亚种NPY基因的DNA和cDNA克隆及序列分析

神经肽Y(Neuropeptide Y,NPY)在机体的摄食活动中发挥重要作用,是哺乳动物最重要的一种内源性促食欲因子。为了解大口黑鲈(Micropterus salmoide)NPY基因的结构及进一步研究该基因在大口黑鲈中的功能作用,采用RT-PCR和RACE技术,克隆了大口黑鲈北方亚种(M.salmoides salmoides)和佛罗里达亚种(M.salmoides floridanus)NPY基因c DNA序列,结果表明两亚种NPY c DNA均包括一个编码99个氨基酸的ORF框和长度为52 bp的5'非编码区(5'-UTR);采用PCR和基因组步移技术获得了长度分别为3 561 bp和3 565 bp的大口黑鲈北方亚种和佛罗里达亚种NPY基因DNA序列。序列分析结果表明,大口黑鲈北方亚种和佛罗里达亚种NPY基因由4个外显子和3个内含子组成。经MATINSPECTOR软件预测,在北方亚种和佛罗里达亚种启动子序列分布有TATA框、CAAT框、CCAAT-Box、GATA-Box等基本转录调控元件。实验在大口黑鲈两亚种NPY DNA序列间发现了6个单个位点碱基差异,与大口黑鲈北方亚种相比佛罗里达亚种启动子区域出现一个4个碱基的插入。不同物种间NPY基因的序列同源性分析表明大口黑鲈与鳜鱼和石斑鱼的NPY基因核苷酸同源性最高,达90%和88%,氨基酸同源性分别为93%和95%。     

大口黑鲈amh基因全长cDNA克隆、表达及多克隆抗体制备

为研究大口黑鲈(Micropterus salmoides)抗缪勒氏管激素(amh)基因的表达及其在性腺发育中的潜在作用,研究利用RACE技术克隆得到了大口黑鲈amh基因,并制备Amh多克隆抗体,通过qRT-PCR、Western Blot分析Amh在大口黑鲈不同组织和不同发育阶段性腺中的表达模式,最后利用HE染色法和免疫组化观察不同发育阶段性腺的形态组织学变化及其与Amh表达的潜在关系。结果显示:大口黑鲈amh基因cDNA序列全长2050 bp,由24 bp5′非编码区、394 bp3′非编码区和1632 bp的开放阅读框组成,共编码543个氨基酸。amh基因mRNA在大口黑鲈11个组织中均有表达,其中雄鱼精巢中表达量最高,肌肉次之,雌鱼卵巢中表达量最高,肌肉次之。amh基因在雌雄鱼不同发育阶段的性腺中表达存在显著差异,精巢中表达量均显著高于卵巢(P<0.05)。同时, Western Blot结果显示Amh蛋白在精巢中表达丰度较高。amh基因在精巢中的表达量呈先上升后下降的趋势,且在孵化后65d鱼精巢中其表达量达到最高(P<0.05),免疫组化结果显示Amh表达于早期精...     

粘虫核糖体蛋白S7 cDNA的克隆和表达分析

运用RT-PCR和RACE技术克隆了粘虫Mythimna separata(Walker)核糖体蛋白S7基因(RPS7)的全长cDNA序列(GenBank登录号:JN582331),并对其进行生物信息学分析。结果表明,粘虫RPS7全长cDNA序列为762 bp,包括5'非编码区32 bp和3'非编码区67 bp。其开放阅读框(573 bp)编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征。该肽链理论分子量为21.924 ku,等电点为9.82,富含4种类型的特定功能位点。该蛋白序列与其他动物RPS蛋白序列具有96.8%～98.2%高度同源性。应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式。RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节。     

家鸽Caveolin-1基因全长cDNA的克隆、序列和组织表达分析

窖蛋白(Caveolin)是一类构成细胞膜上胞膜窖主要结构的标志蛋白,由Caveolin基因家族编码而成。Caveolin-1基因是Caveolin基因家族的成员之一。该实验采用RT-PCR与RACE(rapid amplification of cDNA ends)技术成功地克隆了家鸽Caveolin-1基因的全长cDNA。该cDNA全长2605bp,包含537bp的完整编码区,编码178个氨基酸;分析发现家鸽Caveolin-1基因编码区与牛、家犬、鸡、褐家鼠等核苷酸同源性为80.1%～93.4%,氨基酸同源性高达85.4%～97.2%;半定量RT-PCR分析表明该基因在家鸽各种组织广泛表达,脂肪中表达量最高,各种肌肉中表达量次之,肝脏中表达量最低。此结果说明家鸽Caveolin-1基因可能与脂肪、肌肉中的某些代谢途径有关。     

关岭牛MyoD基因原核表达载体的构建及生物信息学分析

本实验采用RT-PCR方法克隆关岭牛MyoD基因的CDS区,利用Eco RⅠ、XhoⅠ酶对目的片段与pET32a(+)原核表达载体进行酶切,T4连接酶连接过夜,通过转化,测序,构建重组表达载体,并对该基因进行相关生物信息学分析。实验结果获得了关岭牛Myo D基因CDS区,成功构建了p ET-32a(+)-MyoD重组表达载体。MyoD基因编码区为954 bp,编码318个氨基酸,共有31个稀有密码子,表达蛋白大小为34.2 kD。该蛋白为水溶性蛋白,等电点p I=5.8,在体内带负电,二级结构中α-螺旋、无规则卷曲较多。蛋白没有信号肽和明显的跨膜区,为胞内蛋白,主要存在于细胞核中。重组载体表达的Myo D蛋白带有His等标签,蛋白大小为53.6 kD,共497个氨基酸。实验对关岭牛Myo D蛋白的理化性质、结构特点进行了初步分析,以期为研究牛MyoD蛋白特性及其作用机制奠定理论基础。     

斜带石斑鱼2种胰脂肪酶基因全长cDNA序列的克隆与分析

利用RT-PCR和RACE方法克隆得到斜带石斑鱼（Epinephelus coioides）肝胰脏中胆盐活化的胰脂肪酶(bile salt-activated lipase,BSAL)和依赖于辅酶的胰脂肪酶(colipase-dependent pancreatic lipase,PL)基因的全长cDNA序列.BSAL基因全长cDNA序列1 796 bp,编码558个氨基酸,该蛋白序列含有BSAL的全部特征结构区,与其他脊椎动物BSAL的氨基酸序列同源性为49.9%～57.3%.PL基因的全长cDNA序列1 503bp,编码465个氨基酸,该蛋白序列含有PL全部的特征结构区,与其它脊椎动物PL的氨基酸同源性为49.1%～73.9%.系统树分析表明,斜带石斑鱼BSAL和PL与其它物种BSAL、PL和胰脂肪酶相关蛋白(PL-RP)聚于进化树的两个不同分支,属于2种不同的胰脂肪酶.结果证实,在同一鱼类体内也存在BSAL和PL两种胰脂肪酶基因.     

大口黑鲈GHRH-LP和GHRH基因序列同源性、基因结构和时序表达研究

促生长激素释放激素(Growth hormone releasing hormone,GHRH)是生长激素释放调控的重要因子。过去人们一直将鱼类的促生长激素释放激素样多肽(Growth hormone releasing hormone like peptide,GHRH-LP)误认为是GHRH,直到最近才分离出GHRH基因。为了了解GHRH和GHRH-LP基因的结构特征及表达差异,研究克隆了大口黑鲈(Micropterus salmoides)GHRH和GHRH-LP基因,同时应用实时定量PCR技术研究了这两个基因的组织表达及发育时序表达情况。结果显示,大口黑鲈GHRH的成熟肽由27个氨基酸残基组成,GHRH-LP的成熟肽由44个氨基酸残基组成。两个基因均由5个外显子和4个内含子组成,但两者成熟肽编码区在外显子上的分布有明显不同。与其他脊椎动物比较,GHRH同源性为74%100%,而GHRH-LP同源性为41%96%,两者间具有一定的相似性(37%63%)。GHRH基因仅在前脑和延脑中表达,而GHRH-LP基因在中枢神经系统及外周组织中均有表达;在胚胎发育过程中GHRH在神经胚后期检测到表达,其表达水平在仔鱼出膜1d后显著提高,而GHRH-LP在囊胚期及后续发育过程中均检测到表达。基因结构、序列同源性及表达谱研究均表明,GHRH和GHRH-LP存在显著差异,应为具有不同功能的两个基因。       

Failure of low-velocity swimming to enhance recovery from exhaustive exercise in largemouth bass (Micropterus salmoides)

This study was intended to discover whether forcing largemouth bass (Micropterus salmoides) to swim at 0.5 body lengths/second following exercise would expedite recovery relative to fish recovered in static water. Exercise resulted in a suite of physiological disturbances for largemouth bass that included a depletion of anaerobic energy stores, an accumulation of lactate, and increased cardiac output. At 1 h following exercise, exhaustively exercised largemouth bass forced to swim exhibited expedited recovery relative to fish in static water, evidenced by lower concentrations of lactate in white muscle, elevated concentrations of phosphocreatine in white muscle, and reduced concentrations of glucose in plasma. By 4 h postexercise, largemouth bass forced to swim during recovery exhibited signs of physiological disturbance that were absent in fish recovered in static water. These signs of disturbance included a loss of osmotically active particles from plasma, elevated lactate in plasma, reductions of phospocreatine in white muscle, and increased cardiac output. These results are discussed in relation to the body of work with salmonid fishes showing physiological benefits to recovering fish in flowing water.     

Predation of Japanese dace, Tribolodon hakonensis, by largemouth bass, Micropterus salmoides, in experimental aquaria

 To test the size range of prey fish that largemouth bass, Micropterus salmoides, can successfully consume, live Japanese dace, Tribolodon hakonensis, were given as prey fish to individual largemouth bass in aquaria. The ratio of maximum standard length (SL) of the Japanese dace consumed by largemouth bass was 46–69% of bass SL. The maximum length of Japanese dace consumed did not differ significantly between largemouth bass and smallmouth bass (M. dolomieu) previously studied, although largemouth bass have relatively larger mouth sizes than smallmouth bass. Largemouth bass occasionally injured and killed Japanese dace larger than the limit that could be consumed.     

Distribution and diet of Suwannee bass and largemouth bass in the lower Santa Fe River,Florida

Synopsis Suwannee bass,Micropterus notius, and largemouth bass,Micropterus salmoides, were collected by electrofishing in six habitats in the lower Santa Fe River, Florida during May 1981–March 1982. Both bass species were collected concomitantly in all habitats and habitat segregation was not evident. Crayfish (Procambarus spp.) were the primary food of Suwannee bass. Fish were the primary food of largemouth bass, but crayfish were common in the diet of largemouth bass ≥300 mm long. Suwannee bass have a greater throat width and consumed longer and wider forage than did largemouth bass of equal length. Available evidence suggests that Suwannee bass exhibit a positive selection for crayfish and a diverse forage resource, including abundant crayfish, is necessary for a Suwannee bass to coexist with a largemouth bass. This is Journal Series Number 6034 of the Florida Agricultural Experiment Station.     

Phosphoadenylate concentrations and adenylate energy charge of largemouth bass (Micropterus salmoides): relationship with condition factor and blood cortisol

1. Concentrations of phosphoadenylate nucleotides and adenylate energy charge in the dorsal muscle and blood of largemouth bass from a reservoir receiving heated effluent were investigated. These values were compared with the length, weight, body condition and blood cortisol concentrations. 2. Body condition of the largemouth bass ranged from 1043 to 2544, reflecting the range of condition due to starvation of some fish in the population. 3. Blood cortisol concentrations ranged from 4.0 to 23.1 micrograms/100 ml with a mean of 6.3 micrograms/100 ml. 4. The adenylate energy charge of muscle (MATP) and blood ranged from 0.37 to 0.98 and 0.74 to 0.99, respectively. 5. Blood cortisol concentration was positively correlated with body condition but not correlated with the adenylate energy charge of either blood or muscle. 6. Blood cortisol concentration was negatively correlated with concentration of adenylates in both muscle and blood.     

大口黑鲈的卵巢发育周年变化及反季节繁殖研究

文章研究了华中地区池塘养殖大口黑鲈(Micropterus salmoides)卵巢的发育规律, 分析了水温与光照条件变化对卵巢发育的影响, 探究了大口黑鲈反季节繁殖的方法, 旨在充分利用本地区的气候条件, 化劣势为优势, 从根本上解决本地大口黑鲈产业所面临的问题。实验采用形态学、组织学等方法比较分析了大口黑鲈卵巢发育特征, 采用人工控温和人工促熟的方法探究了温度和性激素对大口黑鲈性腺启动发育的影响。研究发现, 华中地区大口黑鲈雌鱼性腺指数(GSI)周年变化在0.63%—7.95%, 10月中旬至12月初水温由20.6℃降至11.0℃期间, 卵巢开始发育至Ⅲ期, 并以Ⅲ期越冬, 至4月中旬繁殖产卵, 5月底结束, 繁殖前约80%的雌鱼绝对繁殖力在4.5万—6.5万粒, 但受水温升高的影响, 卵巢中15%成熟卵母细胞未能产出而逐步退化, 产卵结束时仍有一半以上卵未产出(GSI为4.6%); 雌鱼GSI与肠系膜脂肪系数(MFI)、肝体比(HSI)在10月份至次年4月份呈显著负相关(P<0.05), 表明在此期间, 机体储存的营养物质部分转移至性腺, 优先保证性腺发育。在大口黑鲈反季节繁殖实验中, 采用井水[水温(20±1)℃]降温和控温处理的方法能够促进大口黑鲈性腺的启动发育, 经过3个月处理, 控温组雌鱼卵巢发育至Ⅳ期末, GSI达到4.06%, 而对照组雌鱼卵巢处于Ⅲ期, GSI为2.52%, 两组差异显著(P<0.05), 两组雄鱼精巢均发育至Ⅳ期末, 控温组GSI达0.89%, 对照组为0.73%, 这表明可以通过温度处理来调控大口黑鲈性腺的发育。最后针对反季节繁殖中亲本的培育方法和处理时间等进行了总结, 以期为后续培育反季节大口黑鲈提供依据。     

Experimental trophic ecology of juvenile largemouth bass,Micropterus salmoides,and blue tilapia,Oreochromis aureus

Synopsis The potential for feeding competition between largemouth bass, Micropterus salmoides, and blue tilapia, Oreochromis areus, in Lake Fairfield, Texas was evaluated experimentally. Largemouth bass and blue tilapia were grown in cages alone and in combination with each other. The fish were allowed to feed on the natural food within the lake. Largemouth bass grown in combination with blue tilapia were significantly shorter and weighed less than largemouth bass grown alone. Blue tilapia grown in combination with largemouth bass were statistically significantly longer and heavier than blue tilapia grown alone. Largemouth bass grown alone had diets (volume and number of food items) significantly different than the largemouth bass grown with the blue tilapia. Largemouth bass fed primarily on chironomid larvae and pupae, and odonates, whereas blue tilapia consumed vegetable matter, detritus, and chironomid larvae. Length and weight differences between large-mouth bass grown alone and in combination with blue tilapia, in conjunction with the largemouth bass diet shift, support the theory that these two species compete for food resources.     

A human case of gnathostomiasis nipponica confirmed indirectly by finding infective larvae in leftover largemouth bass meat

A human case of creeping eruption due to Gnathostoma nipponicum was confirmed indirectly by finding infective advanced third-stage larvae in leftover largemouth bass meat. This is the first report indicating that the largemouth bass (Micropterus salmoides) serves as a source of G. nipponicum infection in humans.     

PCR method for detection of largemouth bass virus

A polymerase chain reaction (PCR) method was developed for largemouth bass virus (LMBV). This iridovirus can cause a lethal disease of largemouth bass Micropterus salmoides, but also subclinically infects largemouth bass and other species of fishes. Oligonucleotide primers were designed to specifically amplify the major capsid protein gene of LMBV. The protocol for sample processing and PCR provided a method that was more sensitive than cell culture for detection of LMBV in fish. The specific amplification of LMBV also provided an improved method for confirming the identity of cell-culture isolates presumptively identified as LMBV.     

在低蛋白质饲料中补充必需氨基酸对大口黑鲈生长、体组成和免疫指标的影响

为了研究在低蛋白质饲料中补充晶体必需氨基酸对大口黑鲈生长、体组成和免疫指标的影响,根据鱼体的必需氨基酸组成模式设计了7种等能的试验饲料。其中4种饲料（45CP、40CP、35CP和30CP）的粗蛋白质水平分别为45%、40%、35%和30%,另3种饲料（40AA、35AA和30AA）是在低蛋白质饲料（40CP、35CP和30CP）的基础上添加必需氨基酸,使它们的必需氨基酸含量与45CP（对照组）相一致。用上述饲料对初始体重为（10.13 0.01） g的大口黑鲈进行了89d的饲养试验。饲养试验在室内循环水养殖系统中进行,每种饲料设3个重复,每重复放养30尾鱼。方差分析显示:试验鱼的生长性能、饲料效率、全鱼和肌肉的粗蛋白质含量、成活率以及免疫指标均随着饲料蛋白质含量的降低而显著降低（P0.05）。添加必需氨基酸的35AA和30AA的饲料效率和蛋白质保留率分别显著高于对应的未添加必需氨基酸的35CP和30CP组（P0.05）,但仍显著低于45CP组（P0.05）。40AA的试验鱼血清溶菌酶活性和血清补体活性与45CP组差异不显著（P0.05）。35AA和30AA组的头肾白细胞呼吸爆发活性显著高于35CP和30CP组（P0.05）。30AA组的全鱼粗蛋白质含量以及肥满度显著高于30CP（P0.05）。各组试验鱼的水分和灰分均无显著差异（P0.05）。回归分析显示:在低蛋白质饲料中补充晶体必需氨基酸对大口黑鲈幼鱼的生长、饲料效率和蛋白质保留率所产生的影响与其引起增加了的饲料蛋白质水平而不是饲料的必需氨基酸水平正相关。研究表明,在低蛋白质饲料中补充的晶体必需氨基酸对大口黑鲈的生长、体组成和免疫指标产生的有益作用不及等量的以蛋白质为来源的必需氨基酸。