猪I-FABP基因的分子克隆与组织特异性表达分析

小肠型脂肪酸结合蛋白对长链脂肪酸具有高度的亲和力,参与脂肪酸的吸收和细胞内转运。利用cDNA末端快速扩增（RACE）技术并结合同源克隆策略,克隆到了编码猪小肠型脂肪酸结合蛋白基因（I-FABP）的全长cDNA序列（GenBank接受号：AY960624）,并对系统发育关系等进行了生物信息学分析。猪I-FABP基因的cDNA序列全长614 bp,其中包括399bp的开放式读码框（ORF）,43bp的5’末端非编码区（5’URT）和172bp的3’末端非编码区（3’URT）,编码132个氨基酸残基蛋白,在氨基酸水半上与其他物种的I-FABP具有高度的同源性。以邻接法（Neigbor-Joining,NJ）所构建的系统发育关系表明,猪I-FABP与其他物种的,I-FABP属于同一类群,且与人的遗传距离最近。Northern杂交和半定量RT—PCR分析发现,猪I-FABP在猪体组织中出现约620bp大小的转录本,且在猪体组织中广泛存在,但在小肠组织中表达量最为丰富。     

Molecular characterization of a Ran isoform gene up-regulated in shrimp immunity

Diseases caused by viruses are the greatest challenge to worldwide shrimp aquaculture. Ran gene was an important antiviral gene identified from shrimp and its mRNA level was up-regulated in response to viral infection. In this investigation, a Ran isoform gene (named Ran-iso) cDNA was cloned from shrimp, Marsupenaeus japonicus. The full-length cDNA of Ran-iso was 1286 bp, including a 5′-terminal untranslated region (UTR) of 272 bp, 3′-terminal UTR of 366 bp and an open reading frame (ORF) of 648 bp encoding a polypeptide of 215 amino acids. The deduced protein was highly homologous, it shared 90.64%, 84.19%, 81.48% and 67.58% identities with Ran protein of shrimp, honey bee, human and tobacco respectively. Ran-iso gene was constitutively expressed in 6 tissues examined, including gill, hepatopancreas, hemolymph, heart, intestine and muscle. However, Ran-iso was highest expressed in hepatopancreas (p < 0.01), whereas the expressions of other five tissues were equal and relatively low. Time course analysis showed that the expression level of Ran-iso was obviously up-regulated 2.8 times (at 6 h) as much as that in the control in the hepatopancreas challenged by WSSV. This investigation might provide a clue to elucidate the shrimp innate immunity and would be helpful to shrimp disease control.     

cDNA cloning and the response to overfeeding in the expression of stearoyl-CoA desaturase 1 gene in Landes goose

Stearoyl-CoA desaturase 1 (SCD1) is a rate limiting enzyme in the biosynthesis of monounsaturated fatty acids. It has been cloned from several species: Rattus norvegicus, Mus musculus, Homo Sapiens and Gallus gallus, but not from Anser anser. This study was conducted to isolate the SCD1 cDNA sequence and investigate the effect of overfeeding on SCD1 gene tissue expression in Landes goose. The complete cDNA is 3294 bp in length, with an ORF of 1.083 bp encoding a predicted polypeptide of 360 amino acids and 5′/3′-UTR of 74 and 2137 bp, respectively. Quantitative real time PCR (qPCR) was used to examine SCD1 expression in heart, liver, spleen, lung, kidney, gizzard, glandular stomach, intestine, crureus, pectoral muscle, hypothalamus and adipose tissue (abdominal fat) in both the overfed and control group. SCD1 mRNA was highly expressed in goose fatty liver, and the expression levels of SCD1 in liver and fat of overfeeding group were more than double that of the control group. During the overfeeding period, SCD1 expression in liver and adipose tissue reached the highest level after 70 days, but declined at 79 days. In the control group, after fasting 24 h, the expression level of SCD1 gene in tissues declined sharply. However, SCD1 gene expression in hypothalamus was unaffected. The results of this study provide a theoretical basis to study the relationship between SCD1 gene expression and the formation of fatty liver of Landes goose in response to overfeeding.     

Isolation and characterization of cDNAs encoding Ars2 and Pasha homologues, two components of the RNA interference pathway in Litopenaeus vannamei

The RNA interference (RNAi) is an evolutionarily conserved protective mechanism in eukaryotes against parasitic foreign nucleic acids. Previous studies demonstrated that the RNAi mechanism is important for shrimp antiviral immunity. Here, we report the identification and functional analysis of two key components of the shrimp RNAi activity: Litopenaeus vannamei arsenite resistance gene 2 (LvArs2) and partner of drosha (LvPasha). The full-length cDNA of LvArs2 was 3470 bp, including a 5′ untranslated region (UTR) of 167 bp, a 3′ UTR of 639 bp, and an open reading frame (ORF) of 2664 bp that encoded 887 amino acid residues with an estimated molecular mass of 102.5 kDa. The full-length cDNA of LvPasha was 2654 bp, including a 5′ UTR of 99 bp, a 3′ UTR of 560 bp, and an ORF of 1995 bp that encoded 664 amino acid residues with an estimated molecular mass of 74.2 kDa. Co-immunoprecipitation demonstrated that LvArs2 interacted with L. vannamei Dicer2 (LvDcr2) and LvPasha in Drosophila Schneider 2 (S2) cells, suggesting that LvArs2 may be involved in regulation of the miRNA/siRNA pathways in L.vannamei. Subcellular localization assays demonstrated both LvArs2 and LvPasha proteins mainly presented in the nucleus. After Poly(C-G) stimulation, the expression of LvArs2 was suppressed and expression of LvPasha was enhanced in shrimp gills. These results suggest that LvArs2 and LvPasha may participate in the defense against RNA viruses in crustacea.     

低盐度可诱导鲈鱼胞浆型PEPCK基因表达

磷酸烯醇式丙酮酸羧激酶(PEPCK)催化草酰乙酸生成磷酸烯醇式丙酮酸,是糖异生途径的第1个限速酶.本研究用SMARTRACE技术从鲈鱼肝脏中分离克隆了PEPCK基因的全长cDNA序列.该基因全长2215bp,包含1个123bp的5′非翻译区和217bp的3′非翻译区,开放阅读框为1875bp,编码1个由624个氨基酸组成的蛋白质,该蛋白理论分子量为69.1kD,等电点为5.87.氨基酸序列分析表明,与其它动物的胞浆型PEPCK相似性很高,与黑鲷为94.2%,与大西洋鲑为86.4%,与人为75.9%,而与该鱼线粒体型PEPCK氨基酸同源性只有70.6%.系统发育分析显示,该蛋白首先与其它动物的cPEPCK聚成一支,然后再与鱼类的mPEPCK成簇,认为该PEPCK属于胞浆型.同时用RT-PCR分析了PEPCK基因在10个组织中的表达,结果表明只有在肝脏、消化道和肾脏有较高的表达.将鲈鱼从盐度为25的海水转入盐度为12的海水48h后,肝脏和肾脏的PEPCK基因表达有增加.实验结果表明,本实验克隆的为鲈鱼胞浆型PEPCK,低盐度可诱导其表达.     

Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5′ untranslated region (UTR) of 51 bp, a 3′ UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.     

Hypoxia induces the PDZ domain-containing syntenin in the marine teleost Paralichthys olivaceus

Syntenin is a scaffolding PDZ domain-containing protein with diverse biological activities, including organization of protein complexes in the plasma membrane, regulation of B-cell development, intracellular trafficking, synaptic transmission, and cancer metastasis. In the present study, we isolated and characterized the cDNA of the olive flounder Paralichthys olivaceus syntenin, designated PoSyntenin. The full-length CDS of PoSyntenin with 5′- and 3′-UTR sequences is 2618 bp long and consists of a 909 bp open reading frame preceded by a 161 bp 5′-UTR and followed by a 1551 bp 3′-UTR. The PoSyntenin cDNA encodes a polypeptide of 302 amino acids containing two PDZ domains, which shares 61–80% homology with those of other species, including humans. Expression of the PoSyntenin mRNA was detectable from 1 day post-hatching and constitutively in the brain, spleen, intestine, stomach, eye, liver, kidney, and gill of normal conditioned fish. Expression of the PoSyntenin mRNA was upregulated in the eye, liver, kidney, spleen, brain, gill, and intestine of flounder under hypoxia and was increased by treatment with the hypoxia-mimic CoCl₂ (a HIF-1 inducer) in HINAE cells. Taken together, these results suggest that PoSyntenin is a hypoxia target gene that has a potential role in the hypoxia response mechanism of fish.     

A tandem-repeat galectin involved in innate immune response of the pearl oyster Pinctada fucata

The cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated PfGal) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PfGal was 1386 bp, consisting of a 5′ untranslated region (UTR) of 26 bp, a 3′ UTR of 313 bp, and an open reading frame (ORF) of 1047 bp encoding a polypeptide of 348 amino acids with a predicted molecular weight of 38.09 kDa and theoretical isoelectric point of 8.49. Similar to other tandem-repeat galectins, PfGal contained two tandem carbohydrate recognition domains (CRDs), with typical conserved motifs which were important for carbohydrate recognition, and it appeared to possess neither a signal peptide nor a transmembrane domain. Fluorescent quantitative real-time PCR analyses indicated that PfGal mRNA was highly expressed in hemocytes, digestive gland and mantle, and its expression was increased in all studied tissues after Vibrio alginolyticus challenge. The temporal expression of PfGal mRNA in hemocytes challenged by V. alginolyticus was clearly time-dependent and reached the maximum level at 6 h post-challenge, and then recovered to the original level. These results collectively indicated that PfGal may be involved in the immune response against bacterial infection and clearance of bacterial pathogens in P. fucata.     

Characterization of myosin light chain gene up-regulated in the large yellow croaker immunity by interaction with RanGTPase

RanGTPases are highly conserved in eukaryotes from yeast to human and have been implicated in many aspects of nuclear structure and function. In our previous study, it was revealed that the RanGTPase was up-regulated in large yellow croaker challenged by pathogen. However, the mechanism of RanGTPase in immunity remains unclear. In this investigation, on the basis of protein interaction, it was found that RanGTPase interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. Furthermore, it was found and characterized in this marine fish for the first time. The full-length cDNA of LycMLC was 771 bp, including a 5′-terminal untranslated region (UTR) of 36 bp, 3′-terminal UTR of 279 bp and an open reading frame (ORF) of 456 bp encoding a polypeptide of 151 amino acids. RT-PCR analysis indicated that LycMLC gene was constitutively expressed in the 9 tissues examined, including kidney, liver, gill, muscle, spleen, skin, heart, intestine and blood. The result of quantitative real-time PCR analysis revealed the highest expression in muscle and the weakest expression in skin. Time course analysis showed that LycMLC expression was obviously up-regulated in blood after immunization with either poly I:C or formalin-inactive Gram-negative bacteria Vibrio parahaemolyticus. It indicated that the highest expression was 4.5 times (at 24 h) as much as that in the control (P < 0.05) challenged by poly I:C and 5.0 times (at 24 h) challenged by bacteria. These results suggested that LycMLC might play an important role in large yellow croaker defense against the pathogen infection. Therefore our study revealed a novel pathway concerning immunity of RanGTPase by the direct interaction with the cytoskeleton protein, which would help to better understand the molecular events in immune response against pathogen infection in fish.     

Cloning,characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[α]pyrene exposure

Glutathione S-transferases (GSTs) are phase II enzymes involved in major detoxification reactions of xenobiotics in many organisms. In this study, a full-length cDNA of GST-pi was cloned from the gill of Venerupis philippinarum by rapid amplification of cDNA ends (RACE) method for the first time. The full-length cDNA of V. philippinarum GST-pi (denoted as VpGSTp) was 1142 bp, with a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 438 bp, and an open reading frame (ORF) of 618 bp encoding a protein of 205 amino acid residues with an estimated molecular mass of 23.9 kDa and an predicted isoelectric point (pI) of 7.9. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzyme belongs to the pi-class, and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. Tissue distribution analysis of the VpGSTp mRNA revealed that the GST-pi expression level was observed higher in gill, adductor muscle, mantle and foot while lower in digestive gland. Using quantitative real-time PCR, the dose- and time-related effects of benzo[α]pyrene (B[α]P) on VpGSTp mRNA expression were investigated in gills and digestive gland. The results showed that a time-dependant increase in the expression of VpGSTp was induced by B[α]P and appeared a good linear relationship with B[α]P concentrations. All these results suggested that GST-pi in bivalve had an antioxidant role and VpGSTp expression may be a useful biomarker candidate for monitoring environmental contaminants such as PAHs.     

Molecular cloning, mRNA expression, and characterization of heat shock protein 10 gene from humphead snapper Lutjanus sanguineus

Heat shock protein 10 (HSP10) gene of humphead snapper (Lutjanus sanguineus), designated as ByHSP10, was cloned by rapid amplification of cDNA ends (RACE) techniques with the primers designed from the known EST sequence identified from the subtracted cDNA library of the head kidney of humphead snapper. Sequence analysis showed the full length cDNA of ByHSP10 was 529 bp, containing a 5′ terminal untranslated region (UTR) of 51 bp, a 3′ terminal UTR of 181 bp, and an open reading frame (ORF) of 297 bp encoding a polypeptide of 99 amino acids. Based on the deduced amino acid sequence, the theoretical molecular mass of ByHSP10 was calculated to be 10.92 kDa with an isoelectric point of 9.46. Moreover, chaperonins hsp10/cpn10 signature was found in the amino acids sequence of ByHSP10 by PredictProtein. BLAST analysis revealed that the amino acids of ByHSP10 had the highest homology of 88% compared with other HSP10s. Fluorescent real-time quantitative RT-PCR was used to examine the expression of ByHSP10 gene in eight kinds of tissues of humphead snapper after the challenge with Vibrio harveyi. There was a clear time-dependent expression pattern of ByHSP10 in head kidney, spleen and thymus after bacteria challenge. The expression of mRNA reached the maximum level at the time point of 9 h, 6 h and 24 h, respectively and then returned to control level in 36 h. The up-regulated mRNA expression of ByHSP10 in humphead snapper after bacteria challenge indicated that the HSP10 gene was inducible and might be involved in immune response. A phylogenetic tree was constructed based on the ORF nucleotide sequences of HSP10 for 30 species. The relatonships among them were generally in agreement with the traditional taxonomy which suggested that HSP10 genes could aid in the system classification research.     

Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis

As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149 bp and contained a 168 bp 5′ UTR, a 580 bp 3′ UTR and a 1401 bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P < 0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development.