A cDNA microarray technique applied for analysis of global gene expression profiles in tributyltin-exposed ascidians

To analyze global gene expressions, we constructed a cDNA microarray from a basal chordate, the ascidian Ciona intestinalis. Ciona is a cosmopolitan species and a genomic analysis of Ciona revealed that ascidians had approximately 15,500 protein-coding genes. Our "Ciona intestinalis cDNA chip version 1 (Ci cDNA chip ver. 1)" has arrayed 13,400 unique Ciona cDNAs. To establish a detection system for gene expression profiles in wild ascidians using a cDNA microarray, we analyzed gene expressions in the whole body of Ciona adults after exposure to 100 nM tributyltin (TBT) for 24 h. In our preliminary array data using Ci cDNA chip ver. 1, we found more than 200 genes that showed strong differential expressions. These genes encoded proteins that were concerned with stress response, detoxification, oxidoreduction reaction, biosynthesis, and catabolism. This, the first large cDNA microarray of this animal, should facilitate analyses of global gene expressions following exposure to TBT.     

东海原甲藻cDNA文库构建及尝试性EST分析

东海原甲藻(Prorocentrumdonghaiense)是中国沿海频繁发生的大规模赤潮的原因种之一,大规模分离鉴定东海原甲藻功能基因是理解东海原甲藻赤潮形成过程和机理的重要基础。取对数生长期藻体,经微量总RNA抽提、cDNA合成、cDNA扩增和克隆等步骤,构建了东海原甲藻cDNA文库并进行了尝试性表达序列标签分析。从含有约5000个转化子的文库中随机取150个测序,获得126条EST序列。经网上BlastN及BlastX分析,共发现11个是已知功能的基因的标签。这些基因与东海原甲藻生长发育、物质转换和能量代谢相关。     

青蛤(Cyclina sinensis)cDNA文库构建及免疫相关因子基因的筛选

在鳗弧菌侵染下,利用SMART方法构建了青蛤的cDNA文库,并采用高通量测序方法和BLASTX比对筛选出ESTs及免疫相关因子的基因.结果表明,所构建的cDNA文库的库容量为1.12×106,插入片段均在500bp以上;大于100bp的有效ESTs序列1113条,平均长度725bp,拼接后获得一致性序列420条,其中包括126个叠联群和294个单拷贝EST,BLASTX比对后获得注释序列271条,进一步筛选获得青蛤免疫相关因子基因序列45条,为克隆其免疫防御相关因子的基因提供了重要的基础.     

马氏珠母贝(Pinctada fucata)组织蛋白酶L基因的克隆与表达分析

根据已构建的溶藻弧菌(Vibro alginolyticus)诱导的马氏珠母贝(Pinctada fucata)血淋巴cDNA差减文库得到的ESTs序列, 应用cDNA末端快速扩增(RACE)技术成功克隆了其组织蛋白酶L基因(PFCatL), 并对其进行了生物信息学分析; 应用实时荧光定量PCR (Real-time PCR)技术, 研究了PFCatL基因在溶藻弧菌刺激前后马氏珠母贝足、外套膜、鳃、闭壳肌等8个组织中的表达变化。结果表明, PFCatL基因cDNA全长2004bp, 其中5′非编码区(5′-UTR)50bp, 3′非编码区(3′-UTR)865bp, 开放阅读框(ORF)1089bp, 编码362个氨基酸, 其分子量计算值(MW)为40.52kDa, 理论等电点(IP)为5.20; 生物信息学分析表明, PFCatL含有16个氨基酸残基组成的信号肽序列以及组织蛋白酶前体抑制功能域I29; Clustalw2多重比对发现PFCatL氨基酸序列在催化三联体Cys-His-Asn、底物结合位点以及二硫键形成相关的半胱氨酸残基位点高度保守; Real-time PCR研究发现, PFCatL在马氏珠母贝各组织中均有表达, 但各组织间的表达量存在差异, 其中以肾和闭壳肌中的表达量最高; 溶藻弧菌感染4h后, 外套膜、鳃以及血淋巴中PFCatL基因的表达较感染前显著上调。     

Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5＇-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues （CL to LF）, thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.     

cDNA cloning and expression of a cytochrome P450 1A (CYP1A) gene from the hermaphroditic fish Rivulus marmoratus

We previously described the genomic structure of the cytochrome P450 1A (CYP1A) gene from the hermaphroditic fish Rivulus marmoratus [Kim, I.-C., Kim, Y.J., Yoon, Y.-D, Kawamura, S., Lee, Y.-S., Lee, J.-S., 2004a. Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphroditic fish Rivulus marmoratus and the Japanese medaka Oryzias latipes. Mar. Environ. Res. 58, 125–129]. To further characterize R. marmoratus CYP1A, we cloned the cDNA sequence of a CYP1A gene from this species and also expressed its recombinant protein in an E. coli system. We exposed R. marmoratus to 4-nonylphenol, and found a small induction of CYP1A mRNA in the treated animals. In this paper, we discuss the characteristics of R. marmoratus CYP1A gene as well as its potential use in a biomonitoring assay.     

鲨肝刺激物质类似物基因在大肠杆菌中的表达与产物活性分析

构建了鲨肝刺激物质类似物基因片段的原核表达载体质粒 (pET-sHSS) ,转化大肠杆菌后通过IPTG诱导获得原核表达产物 ,并利用凝胶层析方法对表达产物进行了分离纯化 ;进一步利用MTT比色法研究了该重组产物对肝癌细胞株SMMC -7721的增殖影响。结果表明 ,在1mmol/L的IPTG的诱导下 ,鲨肝刺激物质类似物基因片段在大肠杆菌BL21菌株中获得表达 ,表达产物的相对分子质量大小约为17500u,占菌体可溶性蛋白总量的38%左右 ;纯化后的产物在低浓度下 (<50ng/L)具有明显刺激SMMC7721细胞株增殖的活性 ,高浓度下 (>100ng/L)则明显抑制该肿瘤细胞株的生长 ,与天然鲨肝刺激物质的生物活性类似     

基于形态学与核糖体DNA及其cDNA高通量测序的海洋沉积物中纤毛虫多样性比较

高通量测序技术广泛应用于环境真核微生物多样性的研究,可检获较之传统形态学方法更高的多样性。然而,检获的高分子多样性与基于形态的物种多样性在构成上的差异仍然不明。本研究首次对比分析了基于形态学的南黄海沉积物中的纤毛虫物种多样性与基于核糖体18S DNA和c DNA高通量测序的多样性异同。结果表明:形态鉴定获得8纲、20目、30科、36属共97种纤毛虫;DNA测序检获10纲、28目、55科、76属共174个OTUs;而通过c DNA测序获取的纤毛虫多样性最高,获10纲、31目、68科、99属共284个OTUs。研究发现,形态学方法检获的纤毛虫均为底栖生纤毛虫;两种分子手段检获的多样性更高,群落结构更为近似,覆盖了形态鉴定所获的绝大部分类群。但DNA测序还检获了序列比例高达90%的浮游类群,可能源于包囊或死亡沉降的物种;而c DNA测序检获了约7%的浮游纤毛虫序列。较之DNA测序,c DNA法检获的底栖纤毛虫在群落结构上与形态学结果更为接近。本研究表明,分子手段有助于更全面地揭示沉积物中的纤毛虫多样性,DNA测序可同时揭示休眠包囊、胞外DNA及过去群落的信息,而c DNA测序在研究活动纤毛虫多样性上更有优势。     

宽体沙鳅(Botia reevesae)β-肌动蛋白基因的cDNA克隆与表达分析

利用RT-PCR和快速扩增cDNA末端(rapid amplification of cDNA ends,RACE)技术首次克隆了宽体沙鳅(Botia reevesae)-肌动蛋白基因的cDNA全序列,该序列全长为1795bp,由长100bp的5非翻译区(untranslated region,UTR)、570bp的3非翻译区和1125bp的开放阅读框(open reading frame,ORF)组成,编码375个氨基酸。宽体沙鳅-actin氨基酸序列包含1个糖基化位点、9个N-豆寇酰化位点、3个actin信号位点等主要结构区域。PSI-BLAST比对表明,宽体沙鳅-actin氨基酸与真鲷、罗非鱼、虹鳟等鱼类同源性达99%。NJ法系统进化分析显示宽体沙鳅-actin首先与鲢聚在一起,然后与、尖头等鱼类聚在一起。荧光定量PCR检测-actin基因在宽体沙鳅脑、鳃、心脏、肝、胃等12个组织的表达无显著差异(P<0.05),具有良好的稳定性。     

斜带石斑鱼(Epinephelus coioides)两种催乳素受体基因的 cDNA 克隆和 mRNA 表达分析

采用RT-PCR方法克隆了斜带石斑鱼两种催乳素受体(Prolactin receptor,PRLR)的cDNA序列,序列分析表明:PRLR1开放阅读框为1947bp,共编码649个氨基酸,PRLR2开放阅读框为1749bp,共编码487个氨基酸,PRLR1与PRLR2的氨基酸同源性为40.4%。用Real-time RT-PCR方法研究了PRLR1和PRLR2在各组织和早期发育不同阶段的表达情况,结果表明:PRLR1和PRLR2在所检测的12种组织中均有表达,其中以鳃、肾、肠表达量较高;PRLR1在受精期表达最高,PRLR2在视囊形成期表达最高,而且除受精卵期外PRLR2在各时期的表达量均高于PRLR1。