草鱼成熟脂肪细胞总RNA提取方法研究

获得高质量的RNA是逆转录聚合酶链式反应(RT-PCR)、Northern blot以及转录组测序(RNA-Seq)等分子生物学研究的基础,由于草鱼脂肪细胞中油脂含量较高,从中提取高质量RNA存在困难。本研究以草鱼(Ctenopharyngodon idellus)成熟脂肪细胞为材料,通过增加离心和抽提次数等方式对传统总RNA提取方法进行优化,采用琼脂糖凝胶电泳、核酸定量分析及基因扩增等方法验证所得RNA的完整性、纯度及质量。结果表明,经此方法提取的草鱼成熟脂肪细胞总RNA的A260 nm/A280 nm比值在1.95到2.00之间,28S和18S条带完整,草鱼LPL和β-actin基因扩增条带清晰。认为采用本方法获得的草鱼脂肪细胞RNA样品质量较高,能够用于后续分子生物学研究。     

草鱼PPARα和PPARγ基因的克隆与组织表达差异

根据GenBank上登录的过氧化物酶体增殖物激活受体s(Peroxisome proliferator activated receptor,PPARs)基因序列设计2对引物,提取草鱼肝脏组织总RNA,经RT-PCR扩增获得了PPARα 624bp和PPARγ438 bp.并利用半定量RT-PCR分析T草鱼PPARα和...     

葡萄糖和玉米淀粉对草鱼生长和肠系膜脂肪沉积的影响

采用30%葡萄糖或玉米淀粉作为糖源的配制两种纯化饲料,饲养初始体重为(35.94±1.86)g的两组草鱼,经过为期9周的生长试验,观察葡萄糖和玉米淀粉对草鱼生长和肠系膜脂肪沉积的影响.实验结果显示摄食葡萄糖饲料的草鱼其相对生长率、饲料效率和蛋白质效率均显著高于玉米淀粉组,而肠系膜脂肪占鱼体的百分比在葡萄糖饲料组和淀粉饲料组之间存在显著性差异,分别为1.85%±0.46%和3.56%±0.45%.由此可见,葡萄糖比玉米淀粉对草鱼的生长具有更好的作用,而玉米淀粉比葡萄糖更容易引起草鱼肠系膜脂肪沉积的增加.     

草鱼脂肪细胞提取与保存的研究

无菌条件下取草鱼腹腔脂肪组织,经胶原蛋白酶消化后获取脂肪细胞,通过台盼兰染液染色测定细胞活力以及统计学的方法,对脂肪细胞提取所涉及的消化时间、钢筛规格、离心速度、离心时间等4个重要因素以及脂肪细胞提取后的保存条件进行研究。结果表明,提取草鱼脂肪细胞的最佳条件为:消化时间30min、200目钢筛过滤脂肪细胞液、离心速度为1 000 r/min、离心时间为10 min。草鱼脂肪细胞在室温(25℃)下保存2 d内细胞活率在94.7%以上,而4℃下保存10min后活率已下降至71.1%。     

色泽异常肉的检验与处理

1 黄脂 是脂肪组织中的一种非正常的黄染现象;一般认为与饲料有关,是由于黄色素沉积的缘故. 1.1 感观检查眼观见皮下、网膜、肠系膜、肾周围及腹膜等部位的脂肪均呈深黄色,肌间脂肪组织的着色程度较浅,其他组织通常不着染,脂肪组织除呈现黄色外,无其他变化.黄脂肉尸随放置时间延长而减轻或消失.     

草鱼出血病的病原研究

草鱼出血病的病原研究,始于50年代。1978—84年,分离到一种病原病毒,定名为草鱼呼肠孤病毒或鱼呼肠孤病毒。本文报道从出血病病鱼组织的电镜研究中发现两种病毒颗料,一种即是呼肠孤病毒,另一种是20-30nm大小的病毒。经病毒的核酸分析,前者是双股RNA病毒;后者为单股RNA病毒。用分离到的这两种病毒分别注入1足龄健康草鱼,可发生两类不同症状的出血病;呼肠孤病毒主要导致“肠出血型”症状;另一种病毒(经初步鉴别属于小RNA病毒科病毒)主要导致“肌肉出血型”出血病。由此,可以证实两种病毒都是草鱼出血病的病原病毒,同时也初步解释出现两种不同症状出血病的原因。     

草鱼呼肠孤病毒HZ08株的分离与鉴定

从浙江省湖州地区采集发病草鱼(Ctenopharyngodon idellus)样本中,取症状明显病料的肝、脾、肾组织,经过滤除菌处理后接种草鱼肾脏细胞(CIK).盲传8代,CIK细胞未出现明显细胞病变,但感染细胞固定后经电镜观察发现,细胞质内有大量病毒聚集,病毒无囊膜,近球形,直径约70 nm,形态与已报道的草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)相似.将病毒提纯后分别经DNA酶、RNA酶和绿豆核酸酶消化,证实为双链RNA(dsRNA)病毒.十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示,病毒基因组由11个dsRNA组成,呈现水生呼肠孤病毒基因组典型特征.采用RNA水平3味端加接头的方法获得了S6节段的全长序列,测序结果表明,S6由2 030个核苷酸组成,推测其编码一个分子量约为68.4 kD的蛋白.聚类分析结果显示,该病毒为水生呼肠孤病毒,但在氨基酸水平上与草鱼呼肠孤病毒代表株873株的差异较大,同源性为33%,提示该病毒为一株新型的草鱼呼肠孤病毒.本研究旨在为草鱼出血病防治方法的深入研究奠定基础.     

草鱼ATGL基因的表达及饲喂n-3 HUFAs对其的影响

实验获得了草鱼脂肪组织甘油三酯水解酶(ATGL) 部分cDNA序列(GenBank 登录号为HQ845211), 并进行了序列同源性分析; 采用实时定量反转录聚合酶链式反应(qRT-PCR)方法, 检测了ATGL基因在草鱼不同组织的表达状况; 研究了投喂n-3高不饱和脂肪酸(n-3 HUFAs)对草鱼肝胰脏ATGL基因时序表达的影响。结果显示, 所获得的草鱼ATGL基因部分cDNA序列长度为687 bp, 与人、牛、小鼠、长腭泥虎鱼、大黄鱼等物种的同源性为65%~75%; 该基因在草鱼心脏、肝胰脏、脾脏、鳃、肾脏、肌肉、腹腔脂肪组织、脑、小肠、精巢10个组织中均有表达, 其中在腹腔脂肪组织中表达丰度最高, 在肝胰脏和肌肉中表达丰度次之。处理组草鱼摄食n-3 HUFAs饲料后, ATGL基因的表达水平在第1周和第2周显著高于对照组, 第3周后, 该基因的表达水平在处理组与对照组间无显著差异。研究首次克隆得到草鱼ATGL基因部分cDNA序列, 并发现该基因在草鱼脂质蓄积及代谢较旺盛的组织中表达水平较高, 且其在肝胰脏中的表达受到n-3 HUFAs的影响, 其规律为先被诱导升高, 然后回复到正常水平。     

不同种类淀粉对草鱼生长、肠系膜脂肪沉积和鱼体组成的影响

将试验草鱼随机分配在 12个水族箱中 ,每箱放鱼 30尾 ,分别以玉米淀粉、小麦淀粉、水稻淀粉为糖源配制 3种试验饲料 ,饲养初始体重为 (8.49± 0 .0 4)g的草鱼 80d ,观察不同种类淀粉饲料对草鱼生长、肠系膜脂肪沉积和鱼体组成的影响。每种饲料设 3个平行组 ,日投喂率按鱼体重的 3%计算。试验结果显示 :尽管草鱼对糖的表观消化率以小麦淀粉组最高 ,但摄食小麦淀粉饲料的草鱼与摄食玉米淀粉和水稻淀粉饲料的草鱼在生长上并无显著性差异。然而小麦淀粉组和玉米淀粉组的内脏比、肝胰脏脂肪含量、血浆甘油三酯水平以及肠系膜脂肪占鱼体的百分比显著高于水稻淀粉组 (P <0 .0 5 )。鱼体营养成份组成除了水稻淀粉组全鱼脂肪含量相对低于玉米淀粉组和小麦淀粉组之外 ,其它成份没有太大的差异。     

草鱼HSP90基因cDNA序列克隆及其组织表达差异

根据斑马鱼HSP90(Heat shock protein)序列(NM_131328)设计并合成一对引物,提取草鱼肝脏组织总RNA,RT-PCR扩增获得草鱼HSP90基因cDNA部分序列596 bp,获得GenBank登陆号为FJ517554.将测序结果利用DNAman软件与其他几种已知序列的鱼进行比对,结果表明,草鱼HSP90与斑马鱼、鲤、鲋的同源性依次为90%、89%、92%.同时利用半定量(Semi-quantitative,SQ)RT-PCR技术检测HSP90在草鱼脂肪、肌肉、肠、脑、粘液、性腺、鳔、肝脏、心脏、脾脏、鳃、鳍12个组织的表达差异.结果表明,HSP90在草鱼除了鳍外的其他11个组织中均检测到表达,其中在心脏中表达最高,但与鳃、性腺、脑、脾脏中的表达无显著差异(P>0.05),在脂肪、粘液、鳔组织中的表达显著低于其他几种组织(P<0.05),在肝脏、肌肉与肠中的表达无显著差异(P>0.05).     

刺参组织总RNA提取的研究

利用Trizol法和两种RNA提取试剂盒对刺参的肠、呼吸树、纵肌、体壁等不同组织总RNA进行了提取。综合分析结果表明,F试剂盒提取刺参组织总RNA的效果较其他两种方法而言方法最适宜,能获得纯度高、污染少,而且无降解的高质量总RNA。刺参的肠组织富含RNA,且质地较软容易研磨彻底,是提取总RNA的最佳组织,可获得量大、质优的总RNA。     

Effects of dietary non‐protein energy source levels on growth performance,body composition and lipid metabolism in herbivorous grass carp (Ctenopharyngodon idella Val.)

A study was conducted to determine the effects of dietary non‐protein energy sources on growth, tissue lipid accumulation and lipid metabolism‐related genes expression of grass carp. Triplicate groups of fish were fed for 9 weeks on four isonitrogenous (300 g kg^?1) experimental diets with four levels of non‐protein energy (6.52 kJ g^?1 control diet, 5.32 kJ g^?1 high‐CEL diet, 8.46 kJ g^?1 high‐CHO diet and 8.53 kJ g^?1 high‐LIP diet respectively). Increasing dietary non‐protein energy source levels did not improve the growth, and the high‐CEL diet reduced the growth of grass carp. The high‐CHO diet tended to induce high hepatosomatic index, with high fat and glycogen content of liver. However, the high‐LIP diet caused the high mesenteric fat index, but did not increase liver fat. The mRNA abundance and activities of hepatic lipogenic enzymes were significantly increased in the high‐CHO diet group, whereas the opposite tendencies were observed in the high‐LIP diet group. Peroxisome proliferator‐actived receptor‐α (PPARα) in liver and PPARγ in mesenteric adipose tissue were up‐regulated in the high‐CEL diet group. Lipoprotein lipase (LPL) gene expression was significantly increased both in liver and mesenteric adipose tissue of fish fed the high‐LIP diet, while the LPL gene expression was up‐regulated in liver but down‐regulated in mesenteric adipose tissue of fish fed the high‐CEL diet. These findings suggest that an increase in dietary non‐protein energy sources alters the genes expression of lipid metabolism and increased lipid deposition.     

Development of a modified cortisol extraction procedure for intermediately sized fish not amenable to whole-body or plasma extraction methods

The corticosteroid hormone cortisol is the central mediator of the teleost stress response. Therefore, the accurate quantification of cortisol in teleost fishes is a vital tool for addressing fundamental questions about an animal’s physiological response to environmental stressors. Conventional steroid extraction methods using plasma or whole-body homogenates, however, are inefficient within an intermediate size range of fish that are too small for phlebotomy and too large for whole-body steroid extractions. To assess the potential effects of hatchery-induced stress on survival of fingerling hatchery-reared Spotted Seatrout (Cynoscion nebulosus), we developed a novel extraction procedure for measuring cortisol in intermediately sized fish (50–100 mm in length) that are not amenable to standard cortisol extraction methods. By excising a standardized portion of the caudal peduncle, this tissue extraction procedure allows for a small portion of a larger fish to be sampled for cortisol, while minimizing the potential interference from lipids that may be extracted using whole-body homogenization procedures. Assay precision was comparable to published plasma and whole-body extraction procedures, and cortisol quantification over a wide range of sample dilutions displayed parallelism versus assay standards. Intra-assay  %CV was 8.54 %, and average recovery of spiked samples was 102 %. Also, tissue cortisol levels quantified using this method increase 30 min after handling stress and are significantly correlated with blood values. We conclude that this modified cortisol extraction procedure provides an excellent alternative to plasma and whole-body extraction procedures for intermediately sized fish, and will facilitate the efficient assessment of cortisol in a variety of situations ranging from basic laboratory research to industrial and field-based environmental health applications.     

A more sensitive technique for isolating infectious pancreatic necrosis virus from asymptomatic carrier rainbow trout, Salmo gairdneri Richardson

Abstract. An improved technique for isolating infectious pancreatic necrosis virus (IPNV) is described. It involves trypsinization of the suspect tissue, separation of the trypsinized cells and their co-cultivation with indicator RTG-2 cells. This co-cultivation method is compared with homogenization, followed by membrane filtration, a procedure routinely employed by most disease diagnostic laboratories. From the two IPN-infected farms studied, IPNV could be isolated by homogenization from only 50 and 66%, respectively, of the fish from which virus was detected by co-cultivation. This difference in sensitivity can be explained in two ways, viz: (a) some of the virus is being retained by the membrane filter during filtration to remove contaminants following homogenization; (b) the presence of neutralizing factors in the tissue homogenate are neutralizing the virus and thus no cytopathic effect ensues. No correlation between serum neutralizing antibody levels and the differences in sensitivity of the two methods was found. The co-cultivation method is as practical as, and certainly no more time-consuming than, the homogenization technique.     

Improvement of the extraction procedure for hyaluronan from fish eyeball and the molecular characterization

The object of this study was to improve the isolation procedure of hyaluronan and to compare characteristics of hyaluronan from the eyeball of bigeye tuna Thunnus obesus with other sources. General sources of hyaluronan are from Streptococcus zooepidemicus and rooster comb. Hyaluronan can be also obtained from the vitreous of fish eyes. Pure hyaluronan of higher molecular weight was obtained by the following improved extraction procedure: the frozen vitreous of a tuna eyeball was used to avoid contamination with blood, muscle tissue, and other factors; extracting was carried out over a long time period under cold conditions; cetylpyridinium chloride was used in order to separate mucopolysaccharides containing hyaluronan in the initial procedure without the process of removing fat and protein by reagents. The hyaluronan obtained was characterized by gel permeation chromatography, dynamic light scattering measurements, and viscometry. The characteristics of hyaluronan from tuna eyeballs were similar to those from other sources. However, the viscosity was lower. The possible reason could be ascribed to the wide distribution of molecular size in the vitreous humor of fish eye.     

三角帆蚌外套膜细胞体外培养优化及体内植入培养对细胞生长的影响

以DMEM为基础培养基, 通过优化改良培养基及缓冲液的配方, 对三角帆蚌(Hyriopsis cumingii Lea)外套膜进行细胞培养, 并且以显微观测、RNA/DNA的比值作为该细胞增殖的评价指标, 分别对体外培养细胞的迁出时间、速度、数量以及增殖活力进行测定。分别取体外培养第24、108、120 小时的细胞进行Hoechst DNA荧光标记, 然后将3组标记细胞植入三角帆蚌外套膜中在蚌体内培养, 在植入后第24、72、120、168、216 小时运用RNA/DNA指标检测活体培养的细胞增殖活力。结果表明, 经过优化后的缓冲液和培养基更有利于细胞从外套膜组织中迁出, 迁出速度和细胞数量显著增加(P<0.05), 且细胞的活力随着培养时间的延长而逐渐增大, 培养至108 h时, 细胞活力达到最大, RNA/DNA比值为 24.53, 在108 h后显著下降(P<0.05), 显微观测的细胞生长状况与RNA/ DNA指标测定相吻合。对体外培养的细胞植入蚌体外套膜中再进行培养时发现, 对体外培养活力较好的细胞, 活体内环境可增大细胞的活力, RNA/DNA比值最高达到25.45, 但在活体培养168 h 后活力显著下降(P<0.05); 而对体外培养活力较差的细胞, 活体内环境对细胞活力影响不显著(P>0.05)。本研究旨在为三角帆蚌外套膜细胞增殖的深入研究及建株提供基础性资料。

     

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。