草鱼HSP70基因cDNA部分序列克隆及其组织表达差异

根据鲤热休克蛋白70(Heat shock protein,HSP70)序列(AY120894)设计并合成一对引物,以草鱼(Cteno-pharyngodon idella)肝胰脏组织总RNA为模板,RT-PCR扩增获得草鱼HSP70基因cDNA部分序列,并进行了组织表达差异性研究。结果显示:所获为序列为480 bp,获得GeneBank登陆号为FJ483832。序列测序结果显示,HSP70扩增序列与鲤、斑马鱼、鲋的同源性为:93%、91%、93%。另外,所获序列HSP70在草鱼脂肪、肌肉、肠、脑、粘液、性腺、鳔、肝胰脏、心脏、脾脏、鳃、鳍12个组织的表达存在差异,HSP70在草鱼这12个组织中均检测到表达,其中在鳍中表达最高,极显著高于其他组织(P<0.01);在鳔中表达次之,且与脑、心脏、性腺中表达差异不显著;在粘液中表达最低。     

10种免疫相关基因在斜带石斑鱼组织中的表达分析

采用相对实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)技术,以β-肌动蛋白(β-actin)的表达量作为内参,对健康养殖斜带石斑鱼(Epinephelus coioides)不同组织(头肾、心脏、肝脏、脾脏、鳃、肌肉、鳍、眼、肠道)中的10种免疫相关基因:免疫球蛋白(IgM)、CD4、MHCIIa、TCRβ、CD8a、MHCIa、ISP16、Mx、TNFR14、HSP90的mRNA表达量进行了研究。结果显示,10种免疫相关基因在斜带石斑鱼的10种组织中均有表达。其中体液免疫相关基因[免疫球蛋白(IgM)、CD4和MHCIIa]在鳃中的表达量最高,在其他组织部位的表达量相对较少。细胞免疫相关基因(TCRβ、CD8a和MHCIa)也是在鳃中的表达量最高,在眼、肌肉、肝和肠的部位表达量相对较少。ISP16在鳃和肝中的表达量较高,在肌肉和肠中的表达量较低。Mx在鳃中的表达量较高,在肌肉和肠的表达量较低。TNFR14在眼的表达量较高,在肠的表达量较低。HSP90在鳃和肝中的表达量相对较高,在心和肌肉的表达量较低。     

大口黑鲈糖稳态基因的克隆、分子特征及其营养调控的分析

为进一步研究大口黑鲈(Micropterussalmoides)糖稳态调控特性,本研究克隆了糖稳态调控基因gp1、gp2、pepck1、pepck2、hk、pfk和g6p,分别编码878、842、635、624、918、780和338个氨基酸序列,并进行了进化树、空间分布和禁食对其基因表达影响的分析。多序列比对和进化树分析显示,这些基因与其他脊椎动物的同源序列具有较高相似性。组织表达分析结果显示这些基因主要集中分布于肝脏和肌肉,但呈现出组织表达差异性。其中,gp1在肌肉表达量最高,肝脏次之,在脾脏表达量最低。gp2在肌肉中的mRNA水平也最高,在心脏、肝脏表达也较为丰富。pepck1主要分布在肝脏和肠,在脑、脾脏、肾脏和脂肪中分布较少。pepck2在肝脏和脑中高表达,而在脾脏和鳃中表达量最低。对于hk,其在肝脏、肌肉和心脏的mRNA水平显著高于其余组织。肝脏中g6p的表达也显著高于其余组织。除此之外,其在肌肉、肠和脂肪中分布也较为丰富。pfk的mRNA水平在肝脏和心脏最高,其次是肠和鳃。对实验大口黑鲈禁食后发现,肝脏gp2、pepck2和g6p的表达从禁食第6小时开始上调并持续到24h...     

草鱼孕烷X受体与细胞色素P450 3A mRNA表达相关性初步研究

根据GenBank中斑马鱼(Danio rerio)和虹鳟(Oncorhynchus mykiss)孕烷X受体(pregnane X receptor,PXR)基因序列设计保守区域简并引物,通过PCR扩增获得草鱼(Ctenopharyngodon idellus)PXR cDNA核苷酸序列片段为509 bp,经推导获取169 aa序列。对草鱼与其它物种PXR氨基酸序列进行同源性比较,用以鉴定其在物种中的进化。应用实时荧光定量PCR(qRT-PCR)检测草鱼9种组织PXR和细胞色素P450 3A(cytochrome P450 3A,CYP3A)基因mRNA相对含量,结果表明,其表达丰度依次为:前肠﹥肝脏﹥肾脏﹥鳃﹥肌肉﹥后肠﹥性腺﹥心脏﹥脾脏,CYP3A和PXR基因在草鱼组织中表达分布基本一致,在前肠、肝脏和肾脏高度表达,而在其它组织低度表达。为了进一步研究PXR和CYP3A基因表达相关性,通过细胞,特选用诱导剂利福平(rifampicin,RIF),最佳诱导浓度40μM,孵育1、2、4、6、8、10、12、24 h后,用qRT-PCR检测CYP3A-PXR基因动态表达过程。结果显示,诱导组CYP3A和PXR基因表达量均高于对照组,显示出显著诱导效应。     

红鳍东方鲀3个白介素基因的序列特征及表达分析

本试验分析体质量(190.34±2.13)g的红鳍东方鲀白介素基因IL-1b、IL-8和IL-10的序列特征,检测其在红鳍东方鲀脑、鳃、心脏、肌肉、肝脏和脾脏中的表达,以及每尾红鳍东方鲀注射密度1×10^7 cfu/mL的哈维氏弧菌菌液0.1 mL后0、12、24、48 h在肝脏和脾脏中的表达。试验结果表明,IL-1b、IL-8和IL-10基因的序列全长分别为771、822 bp和844 bp。3个基因在健康成鱼脑、鳃、心脏、肌肉、肝脏、脾脏中均有表达,其中肝脏和脾脏表达显著(P<0.05)。感染哈维氏弧菌后12 h,IL-1b基因在肝脏中显著上调,12 h和48 h在脾脏中显著升高(P<0.05)。感染哈维氏弧菌后12 h和24 h IL-8基因在肝脏和脾脏中均显著上调(P<0.05);而感染后24 h和48 h IL-10基因在肝脏中显著升高,在脾脏中12 h和48 h显著升高(P<0.05)。笔者首次探讨红鳍东方鲀IL-1b、IL-8和IL-10基因的序列特性及表达特征,为研究3个基因的原核表达提供了试验信息,将为进一步深入探究重组蛋白活性及其在红鳍东方鲀健康养殖中的应用奠定基础。     

草鱼碱性氨基酸转运载体y+LAT2 cDNA基因克隆与表达研究

利用逆转录聚合酶链式反应(RT-PCR)和cDNA末端快速扩增(RACE)方法,克隆y+LAT2基因cDNA序列,并利用实时荧光定量PCR探讨y+LAT2在草鱼(Ctenopharyngodon idellus)各组织中的表达情况以及饥饿对其mRNA的影响。结果表明,获得的y+LAT2基因的cDNA序列片段为1849bp,含1371bp的核心序列,编码456个氨基酸。预测草鱼氨基酸序列与斑马鱼(Danio rerio)的同源性高达96.5%,与哺乳动物和两栖动物的同源性在78%～83%,构建的系统进化树与传统形态学分类一致。在草鱼的肌肉、脑、鳃、心、肾脏、肝、后肠、中肠、前肠、脾脏组织均检测到y+LAT2基因的表达。草鱼脾脏、肾脏以及前肠、中肠的y+LAT2 mRNA表达水平对禁食的反应不同,在短期饥饿(14d)期间,其y+LAT2 mRNA表达水平均呈下降趋势。草鱼碱性氨基酸转运载体y+LAT2基因全长cDNA序列的获得,有利于进一步探讨鱼类氨基酸的吸收转运机制。     

白斑综合征病毒感染对凡纳滨对虾热休克蛋白60和90表达的影响

采用实时荧光定量PCR技术分析白斑综合征病毒(WSSV)感染凡纳滨对虾不同时段鳃、肠、肝胰腺、肌肉、类淋巴和血细胞组织中热休克蛋白(HSP)60和90基因mRNA转录水平的变化。结果显示，HSP60和HSP90在这6个组织中都有表达，WSSV感染影响各组织中HSP60和HSP90 mRNA转录水平的表达，WSSV感染抑制HSP60在鳃、肠、肌肉和血细胞的表达，促进HSP90在鳃、肠、肝胰腺、肌肉、血细胞和类淋巴的表达，表明HSP60和HSP90参与WSSV感染免疫反应。     

草鱼Ⅰ型胶原蛋白α1基因cDNA全序列克隆、组织分布及其生物信息学分析

利用PCR和RACE方法首次克隆了编码草鱼肌肉Ⅰ型胶原蛋白的α1基因(COL1A1)的cDNA全长序列,为5772bp,其开放阅读框为4347bp,编码1448个氨基酸。BLAST同源性分析结果显示,草鱼COL1A1基因的氨基酸序列与斑马鱼、金鱼同源性较高,分别为93.90%和93.60%,呈现出较高的保守性。系统进化树分析表明,该基因与斑马鱼、金鱼处于同一支,亲缘性最近。生物信息学分析显示,草鱼COL1A1蛋白质的相对分子质量为137.2ku,理论等电点是5.44,为α螺旋β折叠三重螺旋结构蛋白。有18段三重螺旋重复域,22段低复杂度域,17个功能域。COL1A1蛋白有33～69,1249～1355两个绑定钙的区域和62～104一个绑定锌的区域。利用半定量RT-PCR方法检测的组织表达结果表明,COL1A1基因在草鱼肌肉、肠道、肝胰脏、鳃、皮肤、鳍条、肾脏和脾脏8个组织中均有表达,其中在皮肤、鳃、肾脏、鳍条组织中的mRNA表达量高于其他4个组织(P<0.05)。     

草鱼SREBP-1基因的克隆及糖对其在肝脏中表达的影响

固醇调节元件结合蛋白(SREBPs)是调控糖脂代谢相关基因表达的关键核转录因子。为获知草鱼SREBP-1基因的序列及其在肝脏中的表达规律,本实验采用同源克隆和RACE方法获得了草鱼SREBP-1基因的部分cDNA序列,并通过生物信息学方法对该基因及所编码蛋白的结构特征进行了分析;采用实时荧光定量PCR技术,对SREBP-1基因在8种不同组织的表达规律及低糖(糖含量24%)和高糖(糖含量42%)投喂条件下肝脏中的表达水平进行了研究。结果显示,所克隆到的草鱼SREBP-1基因cDNA长4 760 bp,其中包括开放读码框3 426bp,编码1 141个氨基酸;草鱼SREBP-1具有一个典型的碱性螺旋-环-螺旋亮氨酸拉链结构(bHLH-zip);氨基酸序列比对结果显示,草鱼SREBP-1与其他鱼类的同源性在76%~88%之间,与斑马鱼的进化关系最近;草鱼SREBP-1基因在脑中的表达量最高,肝脏和肠次之,在肾脏、脾脏、肌肉、脂肪和性腺中均有少量表达;与对照组相比,SREBP-1基因在高糖诱导下表达量显著提高(P0.05),低糖诱导下没有显著性差异。研究表明,在高糖负荷条件下,草鱼肝脏中SREBP-1可能会促进糖的利用和转化,从而参与糖代谢调节过程,为丰富鱼类糖代谢调控机理提供研究资料,并有望为提高鱼类对饲料糖的利用效率提供理论依据。     

草鱼NK-lysin基因的克隆、原核表达与活性分析

为研究草鱼抗菌肽NK-lysin基因的组成结构、在组织中的表达差异及其抑菌活性,实验根据已知鱼类NK-lysin基因保守区序列设计上下游引物,采用RT-PCR和RACEPCR技术克隆草鱼NK-lysin基因(Cinkl),RT-PCR进行各组织间的表达分析,并构建Cinkl成熟肽的原核表达载体,采用琼脂糖孔穴扩散法检测重组蛋白的抑菌活性。结果显示,Cinkl c DNA全长768 bp,编码121个氨基酸;基因组DNA全长3 361 bp,包含4个外显子和3个内含子。经多序列比对分析和结构域预测表明,Ci Nkl氨基酸序列具有SAPLIP家族的特征:含有6个保守的半胱氨酸和saposin B蛋白结构域,与斑马鱼Nklc、Nkld的相似性分别为57.98%和63.03%。构建NK-lysin氨基酸序列系统进化树,发现Ci Nkl与斑马鱼Nklc、Nkld聚为一支。RT-PCR检测结果显示,Cinkl m RNA在所有检测的草鱼组织中均有表达,在脾脏中表达量最高,心脏、鳃、中肾和头肾的表达量次之,皮肤、脑、肝脏和肠有微量表达。p ET-28b-MBP与Ci Nkl成熟肽序列构建的原核表达质粒在宿主菌Rosetta(DE3)表达出约57 ku的可溶性融合蛋白,利用Ni-NTA His·Bind树脂进行纯化,Ci Nkl重组蛋白对G–细菌如大肠杆菌M15株、嗜水气单胞菌,以及G+细菌如金黄色葡萄球菌均具有抑菌活性。基于草鱼NK-lysin的组织表达特征和重组蛋白的抑菌活性,推测NK-lysin在草鱼的先天性免疫防御中发挥调节作用。     

Effect of enrofloxacin and emodin on heat-shock proteins’ expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

The aim of the present study was to compare the expression pattern of heat-shock protein 60 (HSP60), 70 (HSP70), and 90 (HSP90) mRNA in hepatic cells of grass carp exposed to enrofloxacin and emodin concentrations. The expression pattern of different genes encoding heat-shock proteins in hepatic cells of grass carp was exposed to graded levels of enrofloxacin (12.5, 25, 50, 100, and 200 μg/ml) or emodin (0.04, 0.2, 1, 5, and 25 μg/ml) for 24 h and were investigated by real-time quantitative PCR. When cells were exposed to up 50 μg/ml enrofloxacin, both HSP60 and HSP70 mRNA levels firstly were increased and thereafter significantly dropped at high concentrations (P < 0.05), while HSP90 decreased with increasing enrofloxacin concentration. Besides, HSP60 and HSP70 expressions were significantly inhibited in the high-concentration groups. In addition, the HSP90 mRNA levels in the treatment exposed to 100 and 200 μg/ml enrofloxacin were significantly lower than those of the control group (P < 0.05). Furthermore, when cells were exposed to graded levels of emodin, HSPs (HSP60, 70, and 90) mRNA levels significantly increased in the groups exposed to 5 and 25 μg/ml of emodin. The different expression pattern of HSPs implied that enrofloxacin could inhibit the expression levels of HSPs, while optimal level of emodin could trigger higher expression levels of HSPs in hepatic cell of grass carp to protect against further damage.     

Molecular characterization of cholecystokinin in grass carp (Ctenopharyngodon idellus): cloning, localization, developmental profile, and effect of fasting and refeeding on expression in the brain and intestine

Cholecystokinin (CCK) is a multi-functional brain–gut peptide in fish and mammals. To investigate the role of CCK in appetite regulation in fish, a 770-bp full-length cDNA sequence of CCK gene was obtained by RT-PCR and rapid amplification of cDNA ends methods in grass carp Ctenopharyngodon idellus. Homology analysis showed that the CCK cDNA sequence of grass carp had the highest similarity (90 %) to that of goldfish Carassius auratus and a higher similarity (>70 %) to those of other teleosts than to mammals. The PCR amplification using genomic DNA identified that the CCK gene of grass carp was comprised of three exons and two introns. Real-time quantitative PCR was used to detect CCK mRNA expression in adult tissues. High levels of gene expression were found in the hypothalamus and pituitary; moderate levels in the intestine, muscle and white adipose tissue; and low levels in other tissues. During early development (i.e., fertilized eggs to 35-day post-hatching larvae) the levels of CCK mRNA expression were higher during embryonic developmental stages than during post-hatch larval stages. Fasting decreased CCK mRNA expression levels in the brain and intestine, whereas refeeding resulted in an increase of expression. The results suggest that CCK mRNA expression has obvious tissue specificity and may have a role in feed intake regulation in grass carp.     

Protective Effect of Emodin against Hyperthermia‐induced Stress in Hepatic Cells of Grass Carp,Ctenopharyngodon idellus

Emodin is an anthraquinone exhibiting several positive benefits of anti‐inflammatory, free radical scavenging, and resistance to stress. The goal of this study is to investigate the effects of emodin on the hepatic cell line of grass carp exposed to hyperthermic stress. Cultured cells were treated with various emodin concentrations (0, 0.04, 0.2, and 1.0 µg/mL) at 27 C for 24 h. Then all cultured cells were exposed to thermal stress by increasing the culture temperature (32 ± 0.5 C) for 0.5 h. Increased temperatures significantly reduced cell viability and increased lactate dehydrogenase (LDH) release in three of the four experimental groups (0, 0.2, and 1 µg/mL emodin) compared with the control. Additionally, reactive oxygen species (ROS) were significantly higher in cells exposed to elevated temperatures and treated with 0.2 or 1 µg/mL emodin and mitochondrial membrane potential was significantly lower in cells treated with 0, 0.2, or 1 µg/mL emodin. Expressions of heat shock proteins (HSP60, HSP70, and HSP90) were significantly higher in all but one of the experimental groups. Our results suggest that 0.04 µg/mL emodin can increase cell viability and HSP90 mRNA level, reduce LDH release and concentration of ROS, and contribute to enhance the resistance to high temperature stress in the hepatic cells of grass carp.     

Effect of heat stress and recovery on viability,oxidative damage,and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

In this study, we investigated the effects of hyperthermia and recovery on cell viability, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA), total antioxidant capacity (T-AOC), and heat shock protein (HSP60, 70, and 90) mRNA expression in the hepatic cells of the grass carp, Ctenopharyngodon idellus. Triplicate groups of cultured cells were exposed to 30, 32, or 34 °C for 0.5 h and then immediately incubated at 27 °C in 5 % CO₂ for 6, 12, 24, or 48 h. Hyperthermia stress greatly reduced cell viability and increased LDH release. Cell damage declined after recovery. Hyperthermia stress increased the lipid peroxide levels and reduced the antioxidant capacity (e.g., reduced SOD and T-AOC) of the cells. However, oxidative damage declined as the recovery period increased, and the levels of MDA, SOD, and T-AOC were restored. After cells were exposed to 32 °C, the expression of HSP60 after recovery for 1, 2, and 4 h (P < 0.05), the expression of HSP70 after recovery for 0.5 and 1 h (P < 0.01), and the expression of HSP90 throughout recovery were significantly higher (P < 0.01) than the prestress levels. During the recovery period, the variations in HSP gene expression reflected the transition period from a state of cellular growth to one of the cellular repairs. In conclusion, hyperthermia depresses cell viability, induces oxidative damage, and increases HSP expression, which plays an important role during hyperthermic stress in grass carp hepatic cells.     

草鱼呼肠孤病毒HZ08株FQ-PCR检测方法的建立及应用

草鱼出血病是危害中国淡水养殖最为重要的病害之一,其病原为草鱼呼肠孤病毒(grass carp hemorrhagevirus,GCRV),其中,HZ08株是当前引起草鱼出血病的主要流行毒株。为建立一种快速、灵敏、特异的草鱼呼肠孤病毒主要流行株检测方法,本研究利用草鱼呼肠孤病毒HZ08株S7基因保守区,设计了一对能特异性扩增154 bp片段的引物和TaqMan探针,用含有S7基因全长的重组质粒PAVX1-S7作为标准品,构建质粒拷贝数与CT值的标准曲线,并对该方法的特异性、可重复性、敏感度进行评价。结果显示:标准曲线在6.0×1010～6.0拷贝数之间有很好的线性关系(r=0.999);实时荧光定量PCR最少可检测到6个阳性质粒,有较高的敏感性;试验内及试验间变异系数分别为0.82%与0.41%～0.52%,重复性强;对水生动物其他病毒均无扩增反应,具有很好的特异性。应用该方法对采集的32份草鱼出血病样品进行检测,其中28份为阳性,而以常规RT-PCR检测同样的样品,仅23份为阳性。本研究建立的草鱼呼肠孤病毒流行株实时荧光定量PCR检测方法在特异性、灵敏度、重复性方面具有较好的测试结果,在GCRV的快速检测和病毒初步定量中应用前景乐观。     

Cloning, tissue distribution and hormonal regulation of stearoyl-CoA desaturase in tilapia, Oreochromis mossambicus

The stearoyl-CoA desaturase cDNA in tilapia (Oreochromis mossambicus) was cloned by RT-PCR and RACE, and it was compared with those in grass carp, common carp and milkfish. Nucleotide sequence analysis revealed that the full length of cDNA (1172 bp) clone encompasses 1008 bp open reading frame (ORF) encoding 336 amino acid residues. The deduced amino acid sequence shares 78–82% identity with the teleosts and 64–66% with mammals compared, and like these fish, the cloned tilapia stearoyl-CoA desaturase amino acid sequence conserves three histidine cluster motifs (one HXXXXH and two HXXHH), which functioned as non-heme iron binding sites, essential for stearoyl-CoA desaturase activity. RT-PCR and Northern blot analysis reveal that tilapia stearoyl-CoA desaturase is expressed only in liver, but the stearoyl-CoA desaturase expression in multiple tissues was observed in milkfish, grass carp and carp. Further, the hormonal regulation of stearoyl-CoA desaturase gene expression was investigated by a single injection of 17β-estradiol and testosterone. The results showed that the administration of 17β-estradiol to tilapia led to a greater increase in desaturase activity than testosterone, and higher doses of steroids produced greater increases in enzyme activity. The comparative RT-PCR analysis showed that the stearoyl-CoA desaturase mRNA level increased significantly in 17β-estradiol treated animals, especially in the groups receiving a single injection of 50 mg 17β-estradiol. This was reflected in the decrease in the saturated fatty acids and the increase in the monounsaturated fatty acids. The proportion of the polyunsaturated fatty acids was not affected.     

草鱼呼肠孤病毒逆转录环介导等温扩增(RT-LAMP)检测方法的建立

根据草鱼呼肠孤病毒(grass carp reovirus,GCRV)衣壳蛋白VP6编码基因的序列设计特异性引物,以病毒全基因组RNA为模板,通过对反应条件进行优化,建立了GCRV的逆转录环介导等温扩增(RT-LAMP)检测方法。检测结果表明,本方法可在63℃下1 h内实现靶片段的大量扩增,扩增产物经凝胶电泳呈现梯型条带,反应体系中添加SYBR Green I荧光染料后,绿色阳性结果明显区别于橙色阴性结果。该检测体系针对草鱼呼肠孤病毒的检测灵敏度高,其最低检测限为33 pg,与常规RT-PCR方法相比较,灵敏度高10倍,且与斑点叉尾鮰呼肠孤病毒(CCRV)、鲤春病毒血症病毒(SVCV)、锦鲤疱疹病毒(KHV)、大鲵虹彩病毒(GSIV)等无交叉反应。该方法灵敏度及特异性高,且不需昂贵仪器设备,为快速检测草鱼呼肠孤病毒与诊断草鱼出血病提供了简捷快速的技术手段。     

草鱼呼肠孤病毒逆转录环介导等温扩增(RT-LAMP)检测方法的建立

根据草鱼呼肠孤病毒 (grass carp reovirus, GCRV)衣壳蛋白VP6编码基因的序列设计特异性引物, 以病毒全基因组RNA为模板, 通过对反应条件进行优化, 建立了GCRV的逆转录环介导等温扩增(RT-LAMP)检测方法。检测结果表明, 本方法可在63℃下1 h内实现靶片段的大量扩增, 扩增产物经凝胶电泳呈现梯型条带, 反应体系中添加SYBR Green I 荧光染料后, 绿色阳性结果明显区别于橙色阴性结果。该检测体系针对草鱼呼肠孤病毒的检测灵敏度高, 其最低检测限为33 pg, 与常规RT-PCR方法相比较, 灵敏度高10倍, 且与斑点叉尾鮰呼肠孤病毒(CCRV)、鲤春病毒血症病毒(SVCV)、锦鲤疱疹病毒(KHV)、大鲵虹彩病毒(GSIV)等无交叉反应。该方法灵敏度及特异性高, 且不需昂贵仪器设备, 为快速检测草鱼呼肠孤病毒与诊断草鱼出血病提供了简捷快速的技术手段。