Identification and expression of the elongator protein 2 (Ajelp2) gene,a novel regeneration-related gene from the sea cucumber Apostichopus japonicus

Elongator proteins comprise six subunits (ELP1–ELP6) and form protein complexes. The elongator protein 2 gene (elp2) encodes a protein with a WD40 repeats domain that acts as a scaffold for complex assembly. It also plays an important role in growth and development. In this study, the full-length cDNA of elongator protein 2 (Ajelp2) was cloned from the sea cucumber Apostichopus japonicus (A. japonicus) using rapid amplification of cDNA ends PCR techniques and comprised 3,058 bp, including a 54 bp 5′ untranslated (UTR), a 526 bp 3′ UTR and a 2,478 bp open reading frame encoding a polypeptide of 825 amino acids. The Ajelp2 sequence showed high homology to 12 other species. The molecular weight and isoelectric of point the presumptive protein were 91.6 kDa and 5.84, respectively. In situ hybridization indicated that the gene is expressed in the body wall, intestine, respiratory tree and longitudinal muscle. The expression level of Ajelp2 increased in recovering of organs in sea cucumber and showed it’s the highest expression level at the 15th day in the intestine and respiratory tree. Its expression then gradually decreased to normal levels. In the body wall, the expression level of Ajelp2 was up-regulated and then down-regulated. These results indicated that Ajelp2 is involved in protein regulation during the regeneration process in the sea cucumber A. japonicus.     

Cloning and expression analysis of the gene encoding fibrinogen-like protein A,a novel regeneration-related protein from Apostichopus japonicus

Fibrinogen-like protein A (FGLA), a member of the fibrinogen-related protein superfamily, exists in different tissues of vertebrates and invertebrates. FGLA plays crucial roles including innate immune response, blood clotting and regeneration. In this study, the fibrinogen-like protein A (fglA) was cloned from Apostichopus japonicus using rapid amplification of cDNA ends PCR techniques. The cDNA sequence of fglA is 1,524 bp with a 849 bp open reading frame encoding a polypeptide of 282 amino acids, with an N-terminal signal peptide and a conserved C-terminal domain. Bioinformatic analysis revealed that the predicted molecular weight of the whole protein is 31.9 kDa and it has an isoelectric point of 5.64. In-situ hybridization demonstrated that fglA is widely distributed in body wall, intestines, longitudinal muscles and respiratory tree. The expression levels of fglA during different regeneration stages in the body wall, intestine and respiratory trees were analyzed by real-time PCR. The expression of fglA gradually increased within 1 h in body wall, and reached a plateau before decreasing to the basal level. This indicates that fglA is associated with the regeneration of Apostichopus japonicus.     

Identification and expression analysis of two Toll-like receptor genes from sea cucumber (Apostichopus japonicus)

Toll-like receptors (TLRs) are a family of type I integral membrane glycoproteins which play pivotal roles in innate immunity. In this study, two TLRs named AjTLR3 and AjToll were cloned from sea cucumber (Apostichopus japonicus). The full-length cDNA sequences of AjTLR3 and AjToll are 3484 bp and 4211 bp, with an open reading frame (ORF) of 2679 bp and 2853 bp, encoding 892 and 950 amino acids, respectively. Both AjTLR3 and AjToll are composed of a leucine-rich repeat (LRR) domain, a transmembrane (TM) domain and an intracellular Toll/interleukin-1 receptor (TIR) domain. Evolution analysis revealed that AjTLR3 and AjToll were clustered with the vertebrate-like TLRs (V-TLRs) and the protostome-like TLRs (P-TLRs), respectively. These two genes were widely expressed in all five tested tissues (body wall, coelomocytes, tube feet, intestine and respiratory tree), but showed different expression patterns. The significantly up-regulated expressions of AjTLR3 and AjToll after peptidoglycan (PGN), lipopolysaccharides (LPS), Zymosan A and polyinosinic–polycytidylic acid (PolyI:C) challenges suggested that they were functionally involved in the immune responses to the Cram-positive bacteria, Gram-negative bacteria, fungi and double-stranded RNA (dsRNA) viruses, respectively.     

Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning,expression and substrate specificity analysis

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3′- and 5′-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (l-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.     

Molecular cloning and sequence analysis of methionine adenosyltransferase from the economic seaweed Undaria pinnatifida

The methionine adenosyltransferase gene (MAT) had been isolated from an economic seaweed Undaria pinnatifida by PCR using degenerate primers. The cDNA was 1,491 bp in length with an open reading frame of 1,194 nucleotides, encoding a deduced protein of 397 amino acids. The protein had a predicted molecular weight of 43.2 kDa, and the isoelectric point was 5.244. The sequence contains a 92 bp 5′-untranslated region (UTR) and a 205 bp 3′-UTR. The methionine adenosyltransferase (MAT) sequence of U. pinnatifida (UpMAT) shared 68–92 % identities with the previous published MAT sequences of other species. Phylogenetic analysis indicated that the phylogenetic relationship of UpMAT with some other seaweeds was closer than with those of higher plants. Under different stress conditions, the relative mRNA expression levels of the MAT of U. pinnatifida (UpMAT) were measured by real-time quantitative PCR, and the results demonstrated that the UpMAT might help to protect the alga against various abiotic stresses.     

Cloning and characterization of hemerythrin gene from Sipuncula Phascolosoma esculenta

Hemerythrin is a non-heme respiratory protein involved in oxygen storage and transport in invertebrates. In the present study, the hemerythrin cDNA was cloned from Phascolosoma esculenta (denoted as PeHr) by PCR and rapid amplification of cDNA ends approaches. The full-length PeHr consisted of 770 bp containing of a 5′-terminal untranslated region (UTR) of 83 bp, a 3′-terminal UTR of 327 bp, and a coding domain sequence of 360 bp encoding a polypeptide of 120 amino acids with estimated molecular mass of 13.6 kDa and theoretical isoelectric point of 5.78. The expression profiles of PeHr were evaluated by real-time RT-PCR under blood loss stress. The expression level of PeHr was significantly up-regulated from 45 to 48 h, then slightly recovered to its original level. The coding sequence of the PeHr was cloned and expressed in Escherichia coli BL21 (DE3) for antibodies preparation. Western blotting analysis conformed that the generated antibodies could specifically identify not only recombinant product, but also native protein from the total protein extraction. Our results indicated that PeHr might be involved into haemocytes regeneration, and its function roles should be further investigated by the generated antibodies.     

An intron-containing,heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae,Diplopoda)

The highly conserved part of the nucleotide-binding domain of the hsp70 gene family was amplified from the soil diplopod Tachypodoiulus niger (Julidae, Diplopoda). Genomic DNA yielded 701, 549 and 540 bp sequences, whereas cDNA from heat shocked animals produced only one distinct fragment of 543 bp. The sequences could be classified as a 70 kDa heat shock protein (hsp70), the corresponding 70 kDa heat shock cognate (hsc70) and a glucose-related hsp70 homologue (grp78). Comparisons of genomic and cDNA sequences of hsc70 identified two introns within the consensus sequence. Generally, stress-70 expression levels were low, which hampered successful RT-PCR and subsequent subcloning. Following experimental heat shock, however, the spliced hsc70 was amplified predominantly, instead of its inducible homologue hsp70. This finding suggests that microevolution in this soil-dwelling arthropod is directed towards low constitutive stress-70 levels and that the capacity for stress-70 induction presumably is limited. hsc70, albeit having introns, apparently is inducible and contributes to the stress-70 response.     

俄罗斯鲟补体C7基因的克隆及表达分析

为研究俄罗斯鲟(Acipenser gueldenstaedti)补体C7基因(AgC7)的功能, 采用RACE (Rapid-amplification of cDNA ends)技术, 获得AgC7的cDNA全长序列为3103 bp, 包括开放阅读框(Open Read Frame, ORF) 2502 bp, 编码833个氨基酸, 5′-UTR (5′ untranslated region)和3′-UTR的长度分别为44和554 bp。同源性分析表明, AgC7与其他鱼类补体C7如斑点雀鳝(Lepisosteus oculatus)、斑点叉尾鲴(Ictalurus punctatus)、斑马鱼(Danio rerio)、大西洋鲑(Salmo salar)、尼罗罗非鱼(Oreochromis niloticus)氨基酸序列的一致性分别为56%、46%、49%、49%、47%, 且都具有TSP1、LDLa、FIMAC和MACPF保守结构域。荧光定量qRT-PCR (quantitative Real-time PCR)结果表明, AgC7在血液、脑、鳃、性腺、心脏、头肾、肠、肝、肌肉、皮肤、脾、胃和后肾组织中均可表达, 且在肠中表达水平最高; 用含有壳寡糖的饲料饲喂俄罗斯鲟60d后, AgC7在这13种组织中表达均有增加, 在肠中增加量最高, 约为对照组的1.51倍; 嗜水气单胞菌(Aeromonas hydrophila)可诱导AgC7, 在血液、鳃、头肾、肠、肝和脾6种组织中瞬时上调表达, 随着病原菌感染时间增加, 基因表达量逐渐降低恢复至正常水平, 其中, 鳃中基因表达量的上调趋势最明显, 最大表达量出现在感染后6h, 为对照组的41.30倍, 12h后基因表达下降并恢复至正常水平, 表明AgC7基因可能参与了俄罗斯鲟抗细菌感染的免疫应答。俄罗斯鲟AgC7具有典型的补体C7基因特征, 且壳寡糖刺激和病原感染后均可引起AgC7基因表达变化, 补体C7可能参与了俄罗斯鲟的免疫应答。     

Molecular Cloning and Analysis of a Cytosolic Hsp70 Gene from <Emphasis Type="Italic">Enteromorpha prolifera</Emphasis> (Ulvophyceae,Chlorophyta)

Heat shock protein 70 (Hsp70) is one of the important members of heat shock protein (Hsp) families and plays essential roles in folding nascent protein, translocation, refolding denatured protein, protein degradation, adverse stress resistance, and so on. In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone full-length cytosolic heat shock protein 70 of Enteromorpha prolifera (designed as EPHsp70). Bioinformatics was used to analyze structural feature, homologous relationship, and phylogenetic position of EPHsp70. The full length of EPHsp70 cDNA was 2,265 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated region of 217 bp, and an open-reading frame of 1,983 bp encoding a polypeptide of 660 amino acids with an estimated molecular weight of 71.39 kDa and an estimated isoelectric point of 5.03. EPHsp70 had five degenerate repeats of tetrapeptide GGMP and three typical Hsp70 signature motifs. The C-terminus amino acid sequence of cytosolic EPHsp70 was EEVD, and the conservation of EPHsp70 of N-terminus was higher than that of C-terminus. The homology between EPHsp70 and the cytosolic Hsp70s of other algae and land plants was more than 70%.