八肋游仆虫Rab家族新成员Eo-rab-1 N基因的克隆与序列分析

Rab蛋白家族属于小分子GTP结合蛋白家族Ras超家族中最大的亚家族,主要在囊泡运输中起作用。实验运用PCR、RT-PCR等技术,从八肋游仆虫中克隆到一种新的rab基因。序列分析结果表明:在大核中,该基因全长884 bp,除去两端的端粒与非编码区,该基因在大核中由723 bp组成。从小核中克隆相应的基因片段,此基因片段序列与大核中序列一致,表明该基因在小核中无内部删除序列的存在。通过RT-PCR,从mRNA获得的该基因的开放读框为663 bp,表明该基因在转录过程中有内含子的删除。大核基因序列和cDNA序

银鲫ZP3的表达特征和卵壳ZP3蛋白的分离

克隆得到银鲫(Carassius auratus gibelio)ZP3基因全长cDNA,在体外表达出银鲫的ZP3融合蛋白,并制备出多抗血清;通过免疫印迹和RT-PCR分析,研究了银鲫ZP3在卵子发生过程中的表达特征和在早期发育胚胎中的状态变化。研究结果表明,银鲫ZP3的转录发生在卵黄发生以前,而ZP3蛋白的翻译起始于卵黄形成阶段,且随着卵母细胞进一步成熟,其含量不断增加。ZP3蛋白的存在状态在受精后和早期胚胎发育过程中发生了明显变化。在受精后5—30min期间,抽提液中原始的ZP3蛋白带迅速消失,取而代

雌核发育银鲫原肠期胚胎和尾芽期胚胎差异表达基因的呈现

构建了雌核发育银鲫原肠期胚胎和尾芽期胚胎间的抑制性差减杂交cDNA质粒文库。对原肠期739个 和尾芽期816个PCR阳性克隆进行斑点杂交,得到72个原肠期和98个尾芽期斑点杂交阳性克隆。测序和基因 数据库比对结果表明:72个原肠期斑点杂交阳性克隆中,包括19个已知基因的cDNA片段和31个没有同源性的 cDNA片段;98个尾芽期斑点杂交阳性克隆中,包括52个已知基因的cDNA片段和37个没有同源性的cDNA片 段。采用虚拟Northern杂交和RT PCR证实了部分基因在银鲫胚胎发育过程中的差异表达。这些

黄鳝DEAD-box家族PL10基因的克隆与序列分析

通过PCR克隆的方法,从黄鳝(Monopterus albus)中得到两个PL10 基因的cDNA片段Mo PL10A和Mo PL10B,长度均为1.127 kb,推测其编码375个氨基酸的蛋白片段.结合其他PL10类同源物序列,对这两条cDNA进行了分析和初步的功能推测.根据此片段的氨基酸序列构建的系统发育树与形态分类结果一致.在不同组织中的RT PCR结果表明Mo PL10A和Mo PL10B的mRNA在各组织中的分布有差异.

鲤鱼两个cGnRH-Ⅱ基因的发现及其在成熟个体的表达分析

促性腺激素释放激素(GnRH)是一个保守的神经十肽家族,在调节脊椎动物的性腺发育和控制性成熟中起至关重要的作用.用RACE和RT-PCR方法,从鲤鱼脑组织克隆得到两个差异的cGnRH-Ⅱ cDNAs序列,其长度分别为622,578 bp.两个cDNA编码的cGnRH-Ⅱ前体均为86个氨基酸,包括一个信号肽、cGnRH-Ⅱ十肽和一个由蛋白水解位点(Gly-Lys-Arg)连接的GnRH相关肽.内含子捕获和Southern杂交证实鲤鱼基因组中有两个cGnRH-Ⅱ编码基因,且两个基因都可能以单拷贝形式存在.鲤鱼

phbB基因在莱茵衣藻中的表达与分子检测

通过构建依赖NADPH的乙酰乙酰CoA还原酶基因(phbB)的衣藻表达载体, 用石英砂VOTEX转化技术, 将phbB基因导入细胞壁缺陷的莱茵衣藻(Chlamydomonas reinhardtii cc-849)中, 用含有10 mg/mL的Zeomycin的平板培养基进行筛选和实验室保持培养, 得到了表达phbB基因的转基因藻株. PCR和Southern blot结果显示phbB基因已整合到莱茵衣藻基因组中. RT-PCR与DNA杂交的检测结果显示, 导入的phbB基因在衣藻中具有转录活性.

纤细裸藻褪色突变株的残留质体及其对核基因表达的影响

用氧氟沙星(Ofl)和链霉素(Sm)分别处理纤细裸藻获得褪色突变株,电子显微镜观察显示细胞中存在残留质体,其中一个Ofl突变株质体有原初类囊体膜形成,而两个Sm突变株质体内有异常致密、发达的膜结构。用PCR方法检查了质体DNA的9个基因,显示所有突变株均有核糖体蛋白基因丢失,其中一个Sm突变株仍保留质体DNA的大部分基因,其余突变株质体DNA则基本丢失。通过差异显示和RT-PCR方法证明叶绿体的退化在完全黑暗异养条件下也可导致某些核基因转录的变化。

鳜免疫球蛋白轻链基因在大肠杆菌中的表达

通过RT-PCR技术从鳜(Siniperca chuatsi)头肾总RNA中获得了免疫球蛋白轻链cDNA,克隆到pGEM-T载体上并测序,测序的2个克隆其可变区cDNA序列相同率为97.3％,属于鳜的同一个轻链可变区基因家族,其变异主要存在于互补性决定区,根据鳜与其他辐鳍鱼类轻链氨基酸序列的多重对准,鳜同鲑(Salmo salar)的可变区的相似性最高(65.7％),而在恒定区,鳜同花狼鳚(Anarhichas minor)的相似性最高(96.3％),都存在特有的连续的丝氨酸残基;鳜同虹鳟(On-corh

外源基因在鱼类核移植胚胎中的转录起始

比较了hGH转植基因在F4代的转MThGH基因鱼, F4代胚胎细胞的核移植后代, 以及F4代尾鳍培养细胞的核移植后代中的转录时序差异. RT-PCR实验结果表明, hGH基因在转基因鱼F4代胚胎中从原肠早期开始转录; F4代胚胎细胞核移植的后代在囊胚早期已能检测到hGH基因转录本; F4代尾鳍培养细胞核移植的后代自16胞期就出现hGH基因的转录．上述结果表明, 鲤鱼卵细胞质对分化细胞核特定基因的再程序化能力是有限的, 进而推论鱼类细胞核移植试验中仅有少部分的供体核能够发生完全再程序化.

Liquid fuel visualization using laser-induced fluoresence during cold start

The presence of liquid fuel inside the engine cylinder is believed to be a strong contributor to the high levels of hydrocarbon emissions from spark ignition (SI) engines during the warm-up period. Quantifying and determining the fate of the liquid fuel that enters the cylinder is the first step in understanding the process of emissions formation. This work uses planar laser induced fluorescence (PLIF) to visualize the liquid fuel present in the cylinder. The fluorescing compounds in indolene, and mixtures of iso-octane with dopants of different boiling points (acetone and 3-pentanone) were used to trace the behavior of different volatility components. Images were taken of three different planes through the engine intersecting the intake valve region. A closed valve fuel injection strategy was used, as this is the strategy most commonly used in practice. Background subtraction and masking were both performed to reduce the effect of any spurious fluorescence. The images were analyzed on both a time and crank angle (CA) basis, showing the time of maximum liquid fuel present in the cylinder and the effect of engine events on the inflow of liquid fuel. The results show details of the liquid fuel distribution as it enters the engine as a function of crankangle degree, volatility and location in the cylinder. A. semi-quantitative analysis based on the integration of the image intensities provides additional information on the temporal distribution of the liquid fuel flow. © 1998 Society of Automotive Engineers, Inc.

应用逆转录聚合酶链反应直接检测草鱼出血病病鱼组织的研究

人工感染GCHV－861后，对处于潜伏期、发病期和恢复期等不同时期的草鱼内脏组织匀浆上清液进行逆转录聚合酶链式反应（RT－PCR）扩增，除恢复期的1条草鱼外，其余样品均得到特异扩增带，而对照组都没有，预示着RT－RCR技术对于草鱼出血病的早期诊断、防治及抗病有种具有重要意义。另外，对于显症出血病草鱼的肝、肾、脾、鳃、肌肉和肠道等组织器官进行检测，结果都为阳性，首次证实了GCHV存在于肝脏中，并对此作了进一步的讨论。

用逆转录聚合酶链式反应检测草鱼出血病病毒的研究

于1994年5月-1995年7月，根据已克隆的草鱼出血病病毒GCHV-861株cDNA的部分序列，设计合成了两对PCR引物，采用RT-PCR技术对GCHV-861及GCHV-873两病毒株的dsRNA进行扩增。结果表明，两对引物仅能特异地检测出GCHV-861病毒株核酸的存在，而不能对GCHV-873病毒株的核酸进行特异扩增，该方法最小可检测出0.1Pg纯化的GCHV-861病毒dsRNA;采用该方法对GCHV-861人工感染的草鱼和稀有鲫组织进行RT-PCR检测，不仅能检测到发病期显症病鱼中GCHV-8

Evolutionary Conservation of Dazl Genomic Organization and its Continuous and Dynamic Distribution Throughout Germline Development in Gynogenetic Gibel Carp

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates. J. Exp. Zool. (Mol. Deu. Euol.) 312B:855-871, 2009. (C) 2009 Wiley-Liss, Inc.

Molecular Characterization and Functional Commonality of Nucleophosmin/Nucleoplasmin in Two Cyprinid Fish

Nucleophosmin/nucleoplasmin has been studied mostly in mammals and amphibians. To clarify the characteristics and function of nucleophosmin/nucleoplasmin in teleost fish, we cloned a full-length cDNA sequence from two cyprinid fish, Carassius auratus gibelio and Carassius auratus. Molecular characterization and multiple sequence alignments suggested that they are the homologs of nucleophosmin. RT-PCR and Western blot detected a specific expression in gonads, and immunofluorescence localization revealed their distribution in oogenic and spermatogenic cells. Furthermore, a sperm decondensation function was demonstrated by immunodepletion and in vitro sperm decondensation experiments. The data suggest that the cloned nucleophosmin should share expressional and functional characterization with nucleoplasmin and therefore provide novel evidence for a functional commonality of nucleophosmin and nucleoplasmin in fish.

Effect of beta-glucan on activity of antioxidant enzymes and Mx gene expression in virus infected grass carp

The effects of beta-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression were studied from grass carp that were challenged with grass carp hemorrhage virus (GCHV). The SOD and CAT activities in erythrocytes and Mx gene expression in spleen from the fish were detected by spectrophotometry and RT-PCR, respectively. Negative control fish were injected with PBS; positive control groups were injected with either P-glucan or GCHV only; and the experimental groups were pre-injected with beta-glucan 15 days prior to injection with GCHV. The results show that the SOD and CAT activities were higher in fish injected with beta-glucan for 15 days than the negative control group injected with PBS. The SOD and CATactivities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with beta-glucan than in infected fish not pre-treated, 15 days after GCHV infection. Mx gene expression levels increased during the early stages (at 12 h and 36 h) of GCHV infection, and it remained at higher levels from the 6th till the 10th day in the beta-glucan pre-treated group, but it was failing from the 6th day in the beta-glucan untreated group. The GCHV-infected group pre-treated with P-glucan had a higher survival rate (60%) than the group not pre-treated with P-glucan (20%), suggesting that beta-glucan possesses or enhances anti-viral responses. (C) 2009 Elsevier Ltd. All rights reserved.