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.

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.

Immunoglobulin joining (J) chain and its expression in the Chinese soft-shelled turtle (Pelodiscus sinensis)

The immunoglobulin (Ig) joining (J) chain plays an important role in the formation of polymeric Igs and their transport into secretions. In the present study, the cDNA sequence of J chain has been cloned from the Chinese soft-shelled turtle (Pelodiscus sinensis) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The cDNA sequence is 2347 bp in length and contains an open reading frame of 480 bp encoding 160 aa including the signal sequence. The deduced amino acid sequence has a high degree of homology with that of an already reported turtle J chain (80.7%), and of chicken (71.3%). By using real-time quantitative RT-PCR analysis, a significant up-regulation of J-chain transcripts was observed in spleen, kidney and blood of turtles injected with inactivated Aeromonas hydrophila, indicating the immune role of J chain in response to bacterial infection. (C) 2009 Elsevier B.V. All rights reserved.

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella)

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases. (C) 2009 Elsevier Ltd. All rights reserved.

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.

Cloning, expression and subcellular distribution of a Rana grylio virus late gene encoding ERV1 homologue

An essential for respiration and viability (ERV1) homologue, 88R, was cloned and characterized from Rana grylio virus (RGV). Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed a highly conserved motif shared by all ERV1 family proteins: Cys-X-X-Cys. RT-PCR and western blot analysis revealed that 88R begins to transcribe and translate at 6 h postinfection (p.i.) and remains detectable at 48 h p.i. during RGV infection course. Furthermore, using drug inhibition analysis by a de novo protein synthesis inhibitor and a viral DNA replication inhibitor, RGV 88R was classified as a late (L) viral gene during the in vitro infection. 88R-EGFP fusion protein was observed in both the cytoplasm and nucleus of pEGFP-N3-88R transfected EPC cells. Although result of immunofluorescence is similar, 88R protein was not detected in viromatrix. Moreover, function of RGV 88R on virus replication were evaluated by RNAi assay. Nevertheless, effect of knockdown of RGV 88R expression on virus replication was not detected in cultured fish cell lines. Collectively, current data indicate that RGV 88R was a late gene of iridovirus encoding protein that distributed both the cytoplasm and nucleus.

Molecular characterization and expression pattern of AFPIV during embryogenesis in gibel carp(Carassiu auratus gibelio)

As a new type of AFPs, AFPIV has been firstly identified in longhorn sculpin (Myoxocephalus octodecimspinosus), and in recent years, its cDNA and amino acid sequence have been reported, and its pancreatic synthesis has been firstly reported in polar fish. However, its expression patterns during fish embryogenesis have not been elucidated yet. By differential screening, we cloned the CagAFPIV in gibel carp, Carassius auratus gibelio, demonstrated its predominant expression during embryogenesis. RT-PCR detection revealed that CagAFPIV was first transcribed from blastula stage and kept a high level during embryogenesis and declined remarkably in hatched larva. In situ hybridization revealed that CagAFPIV transcripts were firstly distributed over the margin and marginal blastomere in blastula stage embryos, at the early-gastrula stage the positive signals distributed in the marginal cells and the internalization cells, and later restricted to the cells the yolk syncytial layer (YSL) from later gastrula stage to larva stage. Consistently, the CagAFPIV protein also kept a high level during embryogenesis, and the high protein level retained some days after the larva hatched. Our work, for the first time, revealed the dynamic expression and distribution of CagAFPIV during embryogenesis.