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Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 （PP1） is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase l（PPlcb）, was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPPlcb, by the rapid amplification of cDNA ends （RACE） technique. The cDNA sequence of SmPPlcb we obtained contains a 984 bp open reading frame （ORF）, flanked by a complete 39 bp 5＇ untranslated region and 462 bp 3＇ untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B （PP2B）. And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPPlcb is extremely conserved in both amino acid and nucleotide acid levels compared with the PPlcb of other vertebrates and invertebrates, and its Kozak motif contained in the 5＇UTR around ATG start codon is GXXAXXGXXATGG, which is different from mammalian in two positions A6 and G3, indicating the possibility of different initiation of translation in turbot, and also the 3＇UTR of SmPPlcb is highly diverse in the sequence similarity and length compared with other animals, especially zebraf＇lsh. The cloning and sequencing of SmPPlcb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot. 相似文献
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Jian Wang Huarong Guo Shicui Zhang Licheng Yin Bin Guo Shaojie Wang 《中国海洋大学学报(英文版)》2008,7(2):184-192
A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 451 bp and an open reading flame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5'UTR of SmTCTP started with a 5'-terminal oligopyrimidine tract (5'-TOP), a typical feature for translationaily controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte-brate TCTPs in a range of 58.8% to 64.1%. The length offish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rer/o) and rohu (Labeo rohita) is 171 aa in length. North-ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves-tigation of the biological function of TCTP in fish. 相似文献
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大口黑鲈抗菌肽hepcidin cDNA序列和结构分析 总被引：2，自引：0，他引：2
以大口黑鲈为材料,提取肝脏总RNA,经RT-PCR扩增出hepcidin cDNA的开放阅读框(ORF)及3′端非编码区序列,应用5′RACE方法得到大口黑鲈hepcidin cDNA5′末端。将所获得的两个片段分别克隆到T载体后进行测序,并拼接成大口黑鲈hepcidin全长cDNA。序列分析表明:大口黑鲈hepcidin全长cDNA为564bp,含有一个258bp的ORF,编码86个氨基酸残基,由信号肽(24个残基)、前肽(42个残基)和成熟肽(20个残基)3部分组成hepcidin前体。在前肽部分具有前肽转化酶典型的RX(K/R)R基元,成熟肽部分含有8个保守的半胱氨酸残基,可形成四个链内二硫桥,使β-折叠结构保持稳定。大口黑鲈Hepcidin与其他鱼类的同源性在29.7%~90.5%间,尤其是信号肽区域,与鳜、尼罗罗非鱼、真鲷、花鲈、黑鯛、金眼狼鲈仅有2~3个氨基酸的差别。 相似文献
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李亚 《广东海洋大学学报》2011,(4)
通过绿僵菌属甘露糖6-磷酸异构酶基因保守核苷酸区域设计简并性引物,采用RT-PCR及RACE-PCR技术成功克隆了金龟子绿僵菌mpi基因cDNA序列。该基因cDNA序列全长为1 513 bp,开放阅读框长度为1 328 bp,共编码441氨基酸。BLAST分析发现该基因演绎的氨基酸序列与其它真菌同源性较高,蛋白结构分析表明MPI蛋白是较保守的蛋白磷酸酶结构特征,主要由α螺旋和不规则卷曲构成。 相似文献
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《中国海洋大学学报(英文版)》2015,(6)
The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B(Bf) serves as the catalytic subunit of complement component 3(C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber(Apostichopus japonicus), termed Aj Bf, was obtained by rapid amplification of c DNA ends(RACE). The full-length c DNA of Aj Bf was 3231 bp in length barring the poly(A) tail. It contained an open reading frame(ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR(5'-terminal untranslated region) and a 384 bp 3'-UTR. Aj Bf was a mosaic protein with six CCP(complement control protein) domains, a VWA(von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of Aj Bf protein was 101 k Da. Quantitative real time PCR(q RT-PCR) analysis indicated that the expression level of Aj Bf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that Aj Bf expressed higher in coelomocytes than in other four tissues. In addation, Aj Bf expression in different tissues was induced significantly after LPS or Poly I:C challenge. These results indicated that Aj Bf plays an important role in immune responses to pathogen infection. 相似文献
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In most bacteria,plants and algae,fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type II fatty acid synthase(FAS II) system.In the FAS II system,enoylacyl carrier protein reductase(ENR) acts as a determinant for completing the cycles of fatty acid elongation.In this study,the cDNA sequence of ENR,designated as IgENR,was isolated from the microalga Isochrysis galbana CCMM5001.RACE(rapid amplification of cDNA ends) was used to isolate the full-length cDNA of IgENR(1 503 bp),which contains an open reading frame(ORF) of 1 044 bp and encodes a protein of 347 amino acids.The genomic DNA sequence of IgENR is interrupted by four introns.The putative amino acid sequence is homologous to the ENRs of seed plants and algae,and they contain common coenzymebinding sites and active site motifs.Under different stress conditions,real-time quantitative polymerase chain reaction(RT-qPCR) showed the expression of IgENR was upregulated by high temperature(35℃),and downregulated by depleted nitrogen(0 mol/L).To clarify the mechanism of lipids accumulating lipids,other genes involved in lipids accumulation should be studied. 相似文献
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The symmetrical body of flatfish larvae changes dramatically into an asymmetrical form after metamorphosis. The molecular mechanisms responsible for this change are poorly understood. As an initial step to clarify these mechanisms, we used representational difference analysis of cDNA for the identification of genes active during metamorphosis in the Japanese flounder, Paralichthys olicaceus. One of the up-regulated genes was identified as creatine kinase muscle type 1 (CK-M1). Sequence analysis of CK-M1 revealed that it spanned 1 708 bp and encoded a protein of 382 amino acids. The overall amino acid sequence of the CK-M1 was highly conserved with those of other organisms. CK-M1 was expressed in adult fish tissues, including skeletal muscle, intestine and gill. Whole mount in-situ hybridization showed that the enhanced expression of CK-M1 expanded from the head to the whole body of larvae as metamorphosis progressed. Quantitative analysis revealed stage-specific high expression of CK-M1 during metamorphosis. The expression level of CK-M1 increased initially and peaked at metamorphosis, decreased afterward, and finally returned to the pre-metamorphosis level. This stage-specific expression pattern suggested strongly that CK-M1 was related to metamorphosis in the Japanese flounder. Its specific role in metamorphosis requires further study. 相似文献
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用RT-PCR和RACE方法,从荷那龙罗非鱼(Oreochromis hornorum)垂体中克隆到生长激素促分泌素受体(GHSR)cDNA全序列。荷那龙罗非鱼GHSR基因具有GHSR-1a与GHSR-1b两个高度保守cDNA序列。GHSR-1a序列全长1 646 bp,包括225 bp的5′非编码区,266 bp的3′非编码区和1 155 bp的开放阅读框,编码384个氨基酸残基,具有7个跨膜结构域结构(transmembrane domains,TM);GHSR-1b序列全长1 877 bp,包括225 bp的5′非编码区,755 bp的3′非编码区和897 bp的开放阅读框,编码298个氨基酸残基,只具有前5个TM,在第6个TM的第4个氨基酸处开始缺失。将GHSR cDNA序列与基因组序列比较发现,这两种cDNA转录本来自同一个基因的不同变体。 相似文献
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GH-IGF-I轴是鱼体体内一个重要的内分泌生理轴,主要调控鱼体的生长发育。用RT-PCR方法从加州鲈脑垂体和肝脏组织中分别扩增出加州鲈GH和IGF-I cDNA,克隆到pMD19 T-Vector上进行序列测定和分析。结果表明:1)加州鲈GH cDNA开放阅读框长为615 bp,编码204个氨基酸,其中信号肽17个氨基酸,成熟肽187个氨基酸。成熟肽中有四个保守的半胱氨酸残基(分别位于69,177,193,202),可形成两对二硫键。加州鲈GH氨基酸序列与蓝太阳鱼、斜带石斑鱼、金头鲷、虹鳟、鲤鱼、斑马鱼相比较,同源性分别为100%、97%、94%、66%、56%、53%。2)加州鲈IGF-I cDNA开放阅读框长为561bp,编码包括信号肽和B、C、A、D、E五个区域的186个氨基酸,形成成熟肽时,信号肽和E区域被切除。成熟蛋白的氨基酸序列与GenBank中已知的河鲈、三角鲂、金头鲷、舌齿鲈、斑马鱼的相比较,结果发现四个区域的保守性有差异,A区和B区保守性较高,C区和D区保守性较差。加州鲈GH和IGF-I cDNA的获得为进一步研究鱼体GH-IGF-I轴对生长发育的调控机制奠定了基础。 相似文献
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【目的】克隆合浦珠母贝(Pinctada fucata)丝氨酸蛋白酶抑制因子pfser1基因,探讨该基因的组织表达及其在天然免疫过程中的作用,以及与生物矿化过程的关系。【方法】通过RACE技术获得pfser1基因的全长,通过生物信息学分析其序列结构特征,利用实时荧光定量PCR方法检测pfser1基因在不同组织中的表达,检测健康合浦珠母贝在被大肠杆菌(Escherichia coli)MG1655刺激后和在贝壳损伤修复实验中pfser1基因表达量的变化。【结果】合浦珠母贝pfser1基因cDNA全长为1240 bp,包含1035 bp的开放阅读框(ORF),编码344个氨基酸,氨基酸序列的功能结构域含有丝氨酸蛋白酶抑制因子Serpin家族保守结构域。pfser1基因在合浦珠母贝各个组织中均有表达,在外套膜边缘膜中表达量最高;大肠杆菌MG1655刺激后,该基因表达量显著升高;在贝壳损伤修复过程中,pfser1基因表达先升高后受到抑制。【结论】pfser1基因所表达的蛋白参与了合浦珠母贝的天然免疫应答过程,并与生物矿化过程有一定关系。 相似文献
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P1 T重组质粒上含有口蹄疫病毒 (FMDV)GD10分离株的p1cDNA片段 ,以此为模板 ,用PCR方法扩增其中的VP1基因 ,获得大小约 6 40bp的片段。该片段用BglⅡ和BstEⅡ酶切消化后克隆至表达载体 pCAMBIA130 5 .2 ,转化EcoliTOP10感受态细胞。重组质粒经PCR、酶切及序列分析 ,证实VP1基因处于CaMV35S启动子控制 ,且读码框正确 相似文献
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A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP),was identified for the first time from the gut cDNA library of Branchiostoma belched. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histocbemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-NIIAmphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri. 相似文献
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五种鲤科鱼类生长激素cDNA的克隆和序列分析 总被引：1，自引：0，他引：1
通过RT-PCR方法,以草鱼、鳙鱼、鲫鱼、鲤鱼、齐口裂腹鱼等5种鲤科重要经济鱼类的垂体总RNA为模板扩增出其生长激素(fish growth hormone,fGH)的完整ORF序列,并克隆到pMD18-T载体上,命名为pMD-1(草鱼)、pMD-2(鳙鱼)、pMD-3(鲫鱼)、pMD-4(鲤鱼)、pMD-5(齐口裂腹鱼)。测序结果显示,ORF序列其长度均为633 bp。序列分析表明,所有ORF序列均以ATG为起始密码,以TAG为终止密码,编码210个氨基酸残基,推导的生长激素前体由22个氨基酸的信号肽和188个氨基酸的成熟肽组成。同源性分析表明,草鱼、鳙鱼、鲤鱼、鲫鱼和齐口裂腹鱼的fGH同源性在93.3%~99.5%之间。 相似文献
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Ubiquitin, a highly conserved stress-related protein, is assigned multiple functions, such as DNA processing, protein degradation, and ribosome synthesis. The Crassostrea hongkongensis ubiquitin gene (designated ChUbL40) was cloned by a combination of suppressive subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The full-length cDNA of ChUbL40 is 496 bp in length, consisting of a 5’ untranslated region (UTR) of 34 bp, a 3’-UTR of 75 bp and an open reading frame of 387 bp encoding a ubiquitin fusion protein of 128 amino acids. Analysis of the amino acid sequence of ChUbL40 reveals that UbL40 is highly conservative during evolution. The expression patterns of ChUbL40 gene in various tissues were examined by real-time PCR. The expression level of ChUbL40 in haemocytes is down-regulated at 4 h and gradually returned to its original level from 6 h to 24 h after Vibrio alginolyticus challenge. Our results suggest that ChUbL40 is ubiquitously expressed and plays an important role in immune defense against bacterial challenge. 相似文献