Cloning, Expression and Characterization of Translationally Controlled Tumor Protein （TCTP） Gene from Flatfish Turbot （Scophthalmus maximus）

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.     

大口黑鲈抗菌肽hepcidin cDNA序列和结构分析

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。     

Molecular Cloning and Expression ofPoIR2, a Novel Gene Involved in Immune Response in Japanese Flounder （Paralichthys olivaceus）

A novel immune-related gene was expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 (P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.     

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and stone flounder <Emphasis Type="Italic">Kareius bicoloratus</Emphasis>

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems. Supported by National Natural Science Foundation of China (No. 30600455) and the National Basic Research Program of China (973 Program, No.2004CB117402)     

金龟子绿僵菌甘露糖6-磷酸异构酶基因cDNA序列的克隆及分析

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。     

Cloning of the cDNA encoding adenosine 5′-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5’-UTR of 78 bp, a 3’-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.     

The characteristics of <Emphasis Type="Italic">vasa</Emphasis> gene from Japanese sea bass (<Emphasis Type="Italic">Lateolabrax japoni</Emphasis>cas) and its response to the external hormones

The RNA helicase Vasa is an important regulator of primordial germ cell development. Its function in mature fish, especially the hormone-related differences in maturing male fish has seldom been documented. In this study, a full length cDNA sequence of the vasa gene was cloned from Japanese sea bass, Lateolabrax japonicas, and it was named jsb-vasa. Homology analysis showed that jsb-vasa was closely related to its teleost homologs. The spatial distribution of jsb-vasa indicated that it was only highly expressed in testis, showing its germ cell-specific expression pattern. During the testicular development cycle, jsb-vasa was highly expressed during early period of spermatogenesis, and reduced when spermatogenesis advanced. In addition, the jsb-vasa gene expression was significantly inhibited at 6 h, 12 h and 24 h after injecting hCG (human chorionic gonadotropin) and GnRHa (Gonadotropin-releasing hormone analogue), indicating that jsb-vasa gene may play an important role in spermatogenesis of Japanese sea bass, and be under the regulation of external sex hormones.     

Characterization and Expression Analysis of a Complement Component Gene in Sea Cucumber (Apostichopus japonicus)

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.     

Construction of a cDNA library for sea cucumber <Emphasis Type="Italic">Acaudina leucoprocta</Emphasis> and differential expression of ferritin peptide

Acaudina leucoprocta is an edible sea cucumber of economic interest that is widely distributed in China. Little information is available concerning the molecular genetics of this species although such knowledge would contribute to a better understanding of the optimal conditions for its aquaculture and its mechanisms of defense against disease. Therefore, we constructed a cDNA library and, based on bioinformatics analysis of the sequences, the functions of 75% of the cDNAs were identified, including those involved in cell structure, energy metabolism, mitochondrial function, and signal transduction pathways. Approximately 25% of genes in the library were unmatched. The gene for A. leucoprocta ferritin was also cloned. The predicted amino-acid sequence of ferritin displayed significant homology with other sea-cucumber counterparts but indicated that it was a new member of the ferritin family. Semiquantitative real-time RT-PCR indicated the highest levels of ferritin mRNA expression in the intestine. A polyclonal antibody of ferritin was also produced. These data provide a set of molecular tools essential for further studies of the functions of ferritin protein in A. leucoprocta.     

Cloning and sequence analysis of a full-length cDNA of SmPPlcb encoding turbot protein phosphatase 1 beta catalytic subunit

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.     

Characterization of defensin gene from abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> and its deduced protein

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals＇ acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides （including six Cys）, molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar （70% in common） to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin （hd-def）, a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.     

Expression and localization of a novel phosducin-like protein from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

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 belcheri. 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 histochemistry 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-N1/Amphi-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. Supported by the Pilot Projects of Knowledge Innovation Project of Chinese Academy of Sciences (No. KZCX2-YW-211-03 and MGE2008KG06)     

荷那龙罗非鱼两种GHSR基因的克隆与序列分析

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。     

合浦珠母贝丝氨酸蛋白酶抑制因子基因pfser1克隆与表达

【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。     

红笛鲷α-珠蛋白和β-珠蛋白基因的克隆及序列分析

应用已知的标签序列通过RACE-PCR和RT-PCR方法成功克隆红笛鲷(Lutjanus sanguineus)α-珠蛋白和β-珠蛋白基因的全长cDNA序列。α-珠蛋白和β-珠蛋白基因的cDNA全长分别为560和563 bp,开放阅读框分别为429和444 bp,各编码143和148个氨基酸,理论相对分子质量分别为15690和16400,理论等电点为7.79和6.61。氨基酸序列BLAST结果表明,红笛鲷α-珠蛋白基因和β-珠蛋白基因编码的氨基酸序列与其他鱼类的相似性分别在59%~79%和55%~80%之间。用邻接法(Neighbor-Joining)构建的α-珠蛋白基因和β-珠蛋白基因的系统发育树表明,红笛鲷与真鲷(Pagrus major)、金头鲷(Sparus aurata)和鰤(Seriola quinqueradiata)等亲缘关系较近。     

加州鲈生长激素和胰岛素样生长因子I cDNA的克隆及序列分析

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。