鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特的关键作用,因而也称为微囊藻毒素去毒酶.从淡水食毒藻鱼类鲢鱼(Hypophthalmichthysmolitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列.序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920bp,其中5′-UTR长74bp,3′-UTR长174bp,编码区长672bp,编码223个氨基酸.应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878bp序列.与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.     

鲢鱼解偶联蛋白2全长cDNA序列的克隆及其组织表达

从淡水食毒藻鱼类鲢鱼(Hypophthalmichthysmolitrix)肝脏,通过简并引物克隆解偶联蛋白2(un-couplingprotein2,UCP2)cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏UCP2cDNA全序列。序列分析结果表明,鲢鱼肝脏UCP2cDNA全长1452bp,其中5′-UTR长337bp,3′-UTR长182bp,编码区933bp,编码310个氨基酸,推测的氨基酸序列包含线粒体内膜载体蛋白3个特征结构及解偶联蛋白(UCPs)的特征序列。对鲢鱼不同组织UCP2的表达调控研究发现,鲢鱼组织UCP2基因在肠道、肝脏、肌肉、脂肪组织均大量表达,而在脑组织表达量较低,这与鲢鱼体内微囊藻毒素在这几个组织的分布完全一致,表明UCP2的功能可能与抑制微囊藻毒素引发过量活性氧(ROS)生成有关。     

鲢鱼解偶联蛋白2全长cDNA序列的克隆及其组织表达

从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏，通过简并引物克隆解偶联蛋白2(uncoupling protein 2，UCP2) cDNA核心序列，应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列，最后通过序列拼接获得鲢鱼肝脏UCP2 cDNA全序列。序列分析结果表明，鲢鱼肝脏UCP2 cDNA全长1 452 bp，其中5′-UTR长337 bp，3′-UTR长182 bp，编码区933 bp，编码310个氨基酸，推测的氨基酸序列包含线粒体内膜载体蛋白3个特征结构及解偶联蛋白(UCPs)的特征序列。对鲢鱼不同组织UCP2的表达调控研究发现，鲢鱼组织UCP2基因在肠道、肝脏、肌肉、脂肪组织均大量表达，而在脑组织表达量较低，这与鲢鱼体内微囊藻毒素在这几个组织的分布完全一致，表明UCP2的功能可能与抑制微囊藻毒素引发过量活性氧(ROS)生成有关。     

罗非鱼微囊藻毒素去毒相关基因克隆与活体表达研究

可溶性谷胱甘肽S-转移酶(Soluble glutathione S-transferase,sGST)催化微囊藻毒素(Microcystins,MCs)与还原型谷胱甘肽(GSH)的加合去毒代谢过程,谷胱甘肽过氧化物酶(Glutathione peroxidase,GPX)为sGST的去毒反应提供GSH,解偶联蛋白2(Uncoupling protein 2,UCP2)则可抑制微囊藻毒素诱发活性氧导致的肝细胞凋亡。本研究从罗非鱼肝脏通过简并引物克隆sGST、GPX与UCP2基因cDNA核心序列,并应用5’RACE和3’RACE技术分别扩增罗非鱼肝脏sGST基因cDNA序列5’末端和3’末端序列而获得其cDNA全序列。罗非鱼肝脏sGST基因cDNA全序列长861 bp,其中5’非翻译区(5-’UTR)为25 bp,3’非翻译区(3-’UTR)为167 bp,开放阅读框(ORF)为669 bp,编码222个氨基酸,包含脊椎动物完整sGST的2个功能域:N-末端功能域(GSH结合位点)和C-末端功能域(底物结合位点)。罗非鱼sGST与真鲷、条石鲷(Oplegnathus fasciatus)、斑马鱼同源性较高,达到64.3%—78.5%,而与人、大鼠、小鼠、牛、猪、鸡差异较大,氨基酸同源性为48.2%—55.9%。罗非鱼肝脏GPX、UCP2基因cDNA核心序列长280 bp、776 bp,分别编码92、258个氨基酸。罗非鱼GPX与条石鲷、虹鳟、斑马鱼、人、大鼠、小鼠、牛、猪GPX同源性均较高,达到69.6%—85.9%。罗非鱼UCP2与真鲷、斑马鱼、鲤鱼、欧洲白鲑(Leuciscus cephalus)、草鱼、人、大鼠、小鼠UCP2同源性更高,达到71.8%—93.8%。通过对罗非鱼(5—8 g)活体腹腔注射亚致死量MC-LR(50μg/kg bwt),发现微囊藻毒素对罗非鱼肝脏sGST基因表达有显著的诱导作用(p<0.05),注射微囊藻毒素24h后sGST基因mRNA表达水平上调80%。注射微囊藻毒素24h后,虽然罗非鱼肝脏GPX与UCP2基因mRNA表达水平亦出现明显的升高趋势,但两者均未出现显著性的变化(p>0.05)。本研究从基因表达调控的角度证实,罗非鱼肝脏sGST在微囊藻毒素去毒过程中可能发挥关键作用,同时也说明罗非鱼肝脏GPX、UCP2基因可能在微囊藻去毒过程中发挥协同作用。     

淡水鱼类微囊藻毒素去毒酶基因的克隆

微囊藻毒素去毒酶在鱼类微囊藻毒素去毒过程中起着关键作用,研究成功克隆鲢鱼、鳙鱼、草鱼、鲫鱼、鳜鱼、罗非鱼等淡水鱼类微囊藻毒素去毒酶基因cDNA核心片段而首次获得这些淡水鱼类微囊藻毒素去毒酶氨基酸序列。鲢鱼、鳙鱼、草鱼、鲫鱼、鳜鱼、罗非鱼微囊藻毒素去毒酶基因与人、小鼠、大鼠、牛、猪、羊的谷胱甘肽S-转移酶基因氨基酸同源性为60%左右,表明淡水鱼类微囊藻毒素去毒酶基因在进化上变异性较大,与其承担微囊藻毒素去毒代谢之特殊功能相适应。     

微囊藻毒素去毒酶转基因模型的构建

根据已克隆的鲢鱼(Hypophthalmichthysmolitrix)微囊藻毒素去毒酶cDNA全序列设计特异引物,利用PCR方法获得鲢鱼微囊藻毒素去毒酶基因编码区,将该编码区与绿色荧光蛋白连接,分别构建融合表达载体pEGFP-N1-sGST和双顺反子表达载体pIRES2-EGFP-sGST。利用脂质体转染法将融合表达载体pEGFP-N1-sGST转染Hela细胞,60h后检测到绿色荧光基因表达;通过显微注射,将双顺反子表达载体pIRES2-EGFP-sGST注入斑马鱼(Daniorerio)受精卵,获得了转鲢鱼微囊藻毒素去毒酶基因斑马鱼,从而构建了微囊藻毒素去毒酶转基因模型。上述2种转基因模型的成功构建为进一步研究鲢鱼、鳙鱼(Aristichthysnobilis)、罗非鱼(Oreochromisnilotica)等淡水鱼类微囊藻毒素去毒酶基因调控元件、去毒分子机理及研发转基因鲢鱼、鳙鱼、罗非鱼等微囊藻毒素高效生物去毒器奠定了基础。     

鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

3种不同食性淡水鱼类谷胱甘肽S-转移酶Pi、Mu、Theta型cDNA序列的克隆分析及表达

鱼类谷胱甘肽S-转移酶(glutathione S-transferase,GST)是鱼类一种重要的Ⅱ相去毒酶,在催化毒素与还原谷胱甘肽(GSH)加合去毒代谢过程中具有关键作用。采用RT-PCR及RACE法,分离、克隆得到草鱼、尼罗罗非鱼pi、mu、theta型GST(GSTpi、GSTmu、GSTtheta)基因、鲢鱼GSTmu、GSTtheta基因的cDNA部分序列并推测各自对应的氨基酸序列。氨基酸序列同源性比较和系统进化分析均表明,鲢鱼、草鱼、尼罗罗非鱼与鱼类GST同源性较高,与哺乳类、鸟类、两栖类GST同源性较低,可能与鱼类GST基因在水环境毒素去毒代谢中承担的特殊功能有关。而不同种鱼类GSTtheta的同源性明显要较GSTpi、GSTmu的同源性低,可能与不同淡水鱼类食性及对毒素耐受性不同有关。用实时荧光定量PCR(RT-PCR)检测三种鱼肝脏中三型GST基因组成型表达水平,发现三种鱼各型之间皆有一定差异,尼罗罗非鱼肝脏整体GSTs基因表达很低,GSTtheta显著低于草鱼(P<0.05),GSTmu显著低于鲢鱼(P<0.05)。本研究为从分子水平上研究不同型谷胱甘肽S-转移酶基因在不同食性淡水鱼类体内代谢去毒过程中的作用提供了基础。     

珊瑚藻R-藻红蛋白γ亚基基因的克隆(英文)

根据珊瑚藻(Corallina afficinalis L.)R-藻红蛋白γ亚基N末端部分氨基酸序列(P83592)设计简并引物,结合RACE方法,扩增获得g亚基的全长cDNA序列。结果表明,序列全长为2308 bp(AY209894),5′非编码区长1203bp,3′非编码区长145 bp,编码区长960 bp,编码320个氨基酸组成的前体,包含71个氨基酸构成的信号肽和249个氨基酸组成的成熟蛋白。成熟蛋白序列内部存在重复序列与前人的报道一致。珊瑚藻亚基cDNA序列不同克隆子的测序结果表明,g亚基cDNA序列存在不同的3′末端,说明该基因可能存在多个拷贝或存在转录后加工。此外,扩增获得g亚基DNA序列(AY308999),比较表明编码区内部没有内含子存在。本文是对珊瑚藻R-藻红蛋白g亚基基因序列的首次报道。     

人HAS3启动子的结构与功能初步分析

透明质酸合酶3（hyaluronan synthase 3,HAS3）,是一个参与透明质酸合成的酶分子,在上皮形成和肿瘤转移等过程中起重要作用.为了进一步研究HAS3基因的转录调控机制,本研究克隆鉴定了HAS3基因的启动子.首先应用5′RACE（rapid amplification of cDNA ends,cDNA末端快速扩增）技术鉴定了HAS3基因的转录起始位点,发现了丰度和转录起始位点不同的两种新的HAS3基因剪接变异体.通过PCR定向克隆策略,构建了覆盖HAS3基因5′端侧翼区起始密码子ATG上游约4.3 kb区域的一系列HAS3基因启动子荧光素酶报告基因重组体.启动子活性分析表明,HAS3基因启动子定位于转录起始位点附近约450 bp的区域内.转录因子结合位点分析表明,HAS3基因启动子缺乏典型的TATA盒,但含有典型的GC盒以及C/EBP等其它潜在的转录因子结合位点. 结果提示,Sp1和C/EBP等转录因子可能参与HAS3基因的转录调控.     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

Molecular cloning of rock bream's (Oplegnathus fasciatus) tumor necrosis factor receptor-associated factor 2 and its role in NF-κB activiation

Tumor necrosis factor receptor-associated factor 2 (TRAF2) acts as a transducer of tumor necrosis factor-α (TNF-α)-triggered cell signals which results in inflammation, cell proliferation and antiapoptotic response. In this study, we have cloned cDNA of rock bream (Oplegnathus fasciatus) TRAF2, and analyzed its function in activation of NF-κB. The full length cDNA of rock bream TRAF2 consisted of 95 bp 5' UTR, 335 bp 3' UTR, and 1563 bp ORF encoding 520 amino acids that contained N-terminal RING-type and TRAF-type zinc finger domains and a C-terminal TRAF domain. The deduced amino acid sequence of rock bream TRAF2 showed more than 75% identity with other fish TRAF2s, and even as high as 56% identity with mouse and human TRAF2 proteins. To know whether the rock bream TRAF2 involves in NF-κB activation, Epithelioma papulosum cyprini (EPC) cells harboring an NF-κB reporting vector were transfected with a vector expressing rock bream TRAF2 or a control empty vector. NF-κB activity of EPC cells was significantly increased by exposure to the rock bream recombinant TNF-α. EPC cells transfected with the vector expressing rock bream TRAF2 showed significantly higher NF-κB activity by stimulation with the recombinant TNF-α than cells transfected with a control empty vector, suggesting the present rock bream TRAF2 acts as a transducer of TNF-α-mediated cell signals that enhance NF-κB activation.     

草鱼和翘嘴鲌fgfrhl-1基因的克隆和表达分析

成纤维细胞生长因子受体同源类似物1(fgfrhl-1)基因是目前仅在鱼类基因组中检测到的fgfr基因家族成员, 该序列在鱼类进化过程中高度保守。为研究fgfrhl-1基因的表达情况和具体的功能, 在亲缘关系较远的草鱼(Ctenopharyngodon idellus)和翘嘴鲌(Culter alburnus Basilewsky)中克隆了fgfrhl-1的cDNA序列, 并通过半定量RT-PCR和冰冻切片原位杂交分析了该基因在成体不同组织中的表达情况。克隆结果的序列分析表明: 草鱼fgfrhl-1 cDNA序列全长为1472 bp, 5′-UTR长213 bp, 3′-UTR长56 bp, 开放阅读框长1203 bp; 翘嘴鲌fgfrhl-1 cDNA序列全长为1886 bp, 5′-UTR长298 bp, 3′-UTR长385 bp, 开放阅读框长1203 bp。在两种鱼类中该基因都编码400个氨基酸, 其预测的氨基酸序列同源性高达95.5%。蛋白二级结构预测表明Fgfrhl-1具有FGFRs家族蛋白的胞内酪氨酸激酶区, 跨膜的螺旋区和胞外配体识别结合区, 但其胞外区比FGFRs缺少了3个免疫球蛋白样结构域。通过RT-PCR方法在两种鱼类的心脏、鳃、肝、脾、尾鳍以及肌肉组织的肌间隔中均检测到了fgfrhl-1表达, 但在肌纤维中均没有检测到其表达。对这两种鱼类的肌肉组织、肝脏和脾脏进行的组织切片原位杂交表明fgfrhl-1只在这些组织和器官的结缔组织及导管中表达, 不在间质细胞结构中表达。这些结果说明: fgfrhl-1的成体组织特异性表达模式在不同鱼类中基本一致, fgfrhl-1在鱼类各组织和器官的结缔组织和导管的细胞中表达, 不在间质细胞中表达。因此, fgfrhl-1可能在鱼类结缔组织及导管分化调控或功能维持中有独特作用。     

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.