鱼类谷胱甘肽转移酶基因cDNA克隆及其序列分析

采用RT-PCR方法,从鲢、鲤、鲫3种鱼类肝脏总RNA中克隆出了谷胱甘肽转移酶Pi型(GST Pi)cDNA序列,推导了其编码的氨基酸序列。3种鱼类GST Pi的ORF全长627bp,编码208个氨基酸。翻译起始密码均为ATG,终止密码子均为TGA。鱼类与哺乳动物、两栖类爪蟾以及节肢动物丝虫之间GST Pi氨基酸序列相似度平均值分别为50%、33%、15%左右。5种鱼类之间的氨基酸序列相似度较大,其中鲤科鱼类之间平均为85%左右。我们以GST Pi为分子标记,用最大简约数法(MP)构建了13个物种的系统进

双链RNA病毒诱导的牙鲆胚胎细胞差减cDNA文库的构建

以紫外线灭活的dsRNA病毒草鱼出血病病毒(GCHV)诱导和模拟诱导的牙鲆胚胎细胞为材料,利用抑制性差减杂交(SSH)技术,成功构建了双链RNA病毒诱导的牙鲆胚胎细胞(FEC)差减cDNA文库。以管家基因αtub lin作为差减指标,经检测,该文库差减效率达210倍,表明经病毒诱导后某些差异表达基因也得到了相应倍数的富集。将获得的cDNA片段连接到pGEM T载体,PCR检测显示差减片段在250bp~2 000bp之间。该差减cDNA文库的构建为从分子水平研究牙鲆培养细胞对dsRNA病毒的免疫反应、以及进

银鲫种系细胞标记分子Vasa:cDNA克隆及其抗体制备(英文)

种系细胞始自胚胎发育早期,是动物生殖及生殖工程的基础。为研究鱼类的种系细胞提供标记分子,我们克隆并鉴定了银鲫的vasacDNA即Cagvasa。CagvasacDNA全长2771碱基(nt),编码的蛋白为银鲫Vasa即CagVasa,全长701个氨基酸(aa)。CagVasa蛋白与已知Vasa蛋白的结构特征一致:在N端有14个RGG重复序列,在C端Vasa所特有的8个功能域俱全。银鲫Vasa与鲤鱼、斑马鱼、陆生脊椎动物和果蝇的Vasa蛋白分别有95%,89%,61%-66%和50%的同源性。卵巢切片的RN

雌核发育银鲫四种孵化酶基因全长cDNA的克隆及其特征分析

用抑制性差减杂交结合SMARTcDNA合成和RACE PCR技术 ,克隆得到雌核发育银鲫 (Carassiusauratusgibelio) 4种孵化酶基因的全长cDNA。 4种孵化酶基因分别被命名为GHE1、GHE2、GHE3、GHE4。序列分析表明 ,4种孵化酶基因cDNA的开放阅读框均为 795bp ,都编码 2 6 5个氨基酸。它们推断的编码氨基酸序列同源性在 79 6～95 1 %之间。种系分析表明 ,GHE1、GHE2、GHE3、GHE4的进化地位位于日本鳗鲡(Anguillajaponica

银鲫两个蛋白合成相关基因全长cDNA的克隆及其特征分析

通过构建雌核发育银鲫心跳期SMARTcDNA质粒文库并从文库中随机挑选克隆测序 ,克隆得到银鲫翻译起始因子 3亚单位 2 (GTIF3 S2 )和翻译延伸因子 1亚单位α(GEF 1α)基因全长cDNA。银鲫翻译起始因子 3亚单位 2基因cDNA全长 12 80bp ,开放阅读框位于 117— 10 91bp之间 ,编码 32 5个氨基酸。其推断的氨基酸序列存在三个WD结构域。该基因在鱼类中为首次报道。银鲫翻译延伸因子 1亚单位alpha基因cDNA全长 1784bp ,开放阅读框位于82— 14 6 7

鱼类培养细胞抗病毒基因差减cDNA文库的构建

紫外线灭活的草鱼出血病病毒 (GCHV)能诱导鲫囊胚培养细胞 (CAB)产生高滴度的干扰素 ,从而诱导宿主细胞基因表达的改变并处于抗病毒状态。提取灭活病毒诱导未经病毒诱导的CAB细胞mRNA ,利用抑制性差减杂交技术 ,成功构建了鱼类培养细胞抗病毒基因差减cDNA文库。以鲫管家基因α tubulin和 β actin作为差减指标 ,检测差减cDNA文库的差减效率分别高达 2 15和 2 7倍 ,表明经过病毒诱导后的细胞中 ,某些差异表达基因的富集效率也接近 2 15倍。鱼类抗病毒基因差减cDNA文库的建立

银鲫3个肌球蛋白轻链基因cDNA的克隆与特征分析

国家自然科学基金 ( 30 130 2 4 0 ) ; 中国科学院知识创新工程重要方向项目 (KSCX2 SW 30 3)资助

梅山猪与长白猪肌肉组织间正反向消减cDNA文库的构建

以梅山猪和长白猪的背最长肌为材料 ,利用抑制性消减杂交技术成功构建了梅山猪与长白猪肌肉组织间正反向消减cDNA文库。以管家基因G3PDH作为消减指标 ,检测梅山猪和长白猪两个消减文库的消减效率分别高达 2 10 和 2 5倍 ,表明某些特异于两种肌肉组织的差异表达基因的富集效率也分别接近 2 10 和 2 5倍。从两个消减文库中分别筛选到了 70 9和 6 73个有效阳性克隆 ,PCR鉴定插入片段长度主要分布于 15 0～ 75 0bp之间。不同猪种肌肉组织间消减cDNA文库的构建为进一步分离和鉴定与猪肌

彩鲫Ran基因全长cDNA的克隆及其组织表达特异性分析

根据Ran的保守区序列设计简并引物 ,结合SMARTcDNA合成及RACE PCR技术 ,克隆到彩鲫(Carassiusauratuscolorvariety)的Ran基因的cDNA全长 ,其编码区全长为 6 4 8bp ,编码 2 15个氨基酸。采用BLAST程序在NCBI数据库中对其进行同源基因搜索 ,结果表明 ,其推测的编码氨基酸序列与斑马鱼和鲑鱼的Ran基因编码的氨基酸序列的同源性分别高达 98%和 97%。还对其编码区全长序列进行了原核表达 ,以经纯化的表达蛋白免疫家兔 ,制备出了具有较高特异性

银鲫肌酸激酶M3-CK cDNA的克隆及其表达特征

用抑制性差减杂交结合SMARTcDNA合成和RACE PCR技术克隆到雌核发育银鲫 (Carassiusauratusgibelio)肌酸激酶M 3 CK基因的全长cDNA。银鲫M 3 CKcDNA全长 15 5 1bp ,编码 380个氨基酸 ,与普通鲤鱼(Cyprinuscarpio)M3 CK的氨基酸序列同源性高达 95 %。种系分析表明 ,银鲫M3 CK与其它脊椎动物的肌肉型肌酸激酶聚为较近的一支 ,与鲤鱼的M 3 CK聚在一起 ,与脑特异型肌酸激酶及线粒体型肌酸激酶分歧较大。虚拟Northern

人工合成草鱼生长激素cDNA在大肠杆菌中的表达

经密码子优化的人工合成草鱼生长激素ｃＤＮＡ与表达载体ｐＥＴ－２８ａ（＋）重组，构建重组表达质粒ｐＥＴ－ＧＨ。转化大肠杆菌ＢＬ２１（ＤＥ３），筛选阳性克隆，ＩＰＴＧ诱导表达。１２．５％的ＳＤＳ－ＰＡＧＥ分析显示，大肠杆菌表达产物中含有与草鱼生长激素分子量一致的新增蛋白带，激光密度扫描，其产量约占菌体总蛋白的４０％。金属离子螯合层析柱亲和纯化，获得电泳纯的重组蛋白。Ｗｅｓｔｅｒｎ－ｂｌｏｔｔｉｎｇ和酶联免疫吸附受体法检测证实：重组蛋白与抗草鱼生长激素的多克隆抗体发生特异性结合；复性后的重组蛋白有与天然草鱼生长

银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A_1的cDNA克隆

分别取行天然雌核发育繁殖的银鲫和两性生殖的彩鲫的卵母细胞为材料 ,提取总RNA ,分离mRNA ,进而反转录合成cDNA并定向插入λgtllSfi Not克隆载体 ,经体外包装构建了银鲫与彩鲫卵母细胞的表达型cDNA文库。测试结果表明库容量分别达到 3 1× 1 0 6(银鲫 )和 1 6× 1 0 6(彩鲫 )。进一步人工合成CyclinA1 保守引物 ,采用PCR扩增文库的方法 ,克隆了银鲫 (1 61 6bp)与彩鲫 (1 62 6bp)的CyclinA1 全长cDNA。序列分析结果表明 :两种鱼编

鲑鱼胰岛素样生长因子I cDNA在镜鲤中的表达

通过显微注射技术，将冠以鲤鱼β－肌动蛋白基因（ＣＡ）启动调控顺序的鲑鱼胰岛素样生长因子Ｉ（ｓＩＧＦ－Ｉ）的ｃＤＮＡ，即ＣＡｓＩＧＦｃ，注入镜鲤（Ｃｙｐｒｉｎｕｓ　　ｃａｐｉｏｖａｒ．　　ｓｐｅｃｔｕｌａｒｉｓ）受精卵内。经聚合酶链式反应（ＰＣＲ）技术检测，受体胚胎发育各期均携带有外源基因拷贝。在由此发育的５月龄鱼中，８０％的个体带有外源基因拷贝。用逆转录一聚合酶链式反应（ＲＴ－ＰＣＲ）技术检测，发现２／３以上的受体胚胎具有表达外源基因的能力。在５月龄的转基因镜鲤中，只有部分组织和器官可检测到外源基因的存在

Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization

The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution. (C) 2008 Published by Elsevier Inc.

Identification of differential transcript profiles between mutual crossbred embryos of zebrafish (Danio rerio) and Chinese rare minnow (Gobiocypris rarus) by cDNA-AFLP

The crosstalk between naive nucleus and maternal factors deposited in egg cytoplasm before zygotic genome activation is crucial for early development. In this study, we utilized two laboratory fishes, zebrafish (Danio rerio) and Chinese rare minnow and Chinese rare minnow (Gobiocypris rarus), to obtain mutual crossbred embroys and examine the interaction between nucleus and egg cytoplasm from different species. Although these two types of crossbred embryos originated from common nuclei, various developmental capacities were gained due to different origins of the egg cytoplasm. Using cDNA amplified fragment length polymorphism (cDNA-AFLP), We Compared transcript profiles between the mutual crossbred embryos at two developmental stages (50%- and 90%-epiholy). Three thousand cDNA fragments were generated in four cDNA pools with 64 primer combinations. All differently displayed transcript-derived fragments (TDFs) were screened by (lot blot hybridization, and the selected sequences were further analyzed by semi-quantitative RT-PCR and quantitative real-time RT-PCR. Compared with ZR embryos, 12 genes were up-regulated and 12 were down-regulated in RZ embryos. The gene fragments were sequenced and subjected to BLASTN analysis. The sequences encoded various proteins which functioned at various levels of proliferation, growth, and development. One gene (ZR6), dramatically down-regulated in RZ embryos, was chosen for loss-of-function study; the knockdown of ZR6 gave rise to the phenotype resembling that of RZ embryos. (c) 2008 Elsevier Inc. All rights reserved.