| Abalone Haliotis discus hannai Ino are large algivorous marine gastropods, and the most commercially important specie of gastropods in aquaculture for Asia. However, abalone culture has suffered serious problems of mortality from disease outbreaks, environmental contamination and decreased innate immunity of abalone in China and other countries or districts. It is an emergent and necessary work for us to explore the immune system of abalone and to find effective methods to control these diseases or to allieveate stresses. It has been proved that a mass of reactive oxygen species (ROS) could be induced by pathogens, malnutrition, chemicals, radiation, high or cold temperature, ressloved oxygen and other possible stresses. Though ROS can kill foreign invaders, the excessive accumulation of these toxic by-products could cause serious damage to lipids, proteins and nucleic acids. In order to protect cells against the toxicity caused by ROS, animals have evolved protective antioxidant systems, including antioxidant enzymatic systems such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), and non-enzymatic systems, such as vitamin C and glutathione, etc.It is well known that all the materials and energy are rooted in nutrients used for the production, elimination, usage, damnification and rehabilitation of ROS.Namely, all components are directly or indirectly resulted from nutrients or antioxidants in the food. Therefore, it is important for optimal nutrition states to well balance the homeostasis between production and elimination of ROS,maintain the reduction states cellular environment, and induce normal function of ROS and each other. In addition, many studies have confirmed that animals could maintain better homeostasis of oxidation/reduction through nutrition regulation with adequate nutrients, including selenium, zinc, iron and vitamin C, etc.However, there has no published literature on the interaction mechanisms between dietary minerals and anti-oxidative responses at gene transcriptional levels in abalone Haliotis discus hannai Ino.In addition, identification and cloning of the genes involved in anti-oxidative response will help us to understand the molecular basis of the innate immune response to the malnutrition stresses.In the present study, selenium-dependent glutathione peroxidase(Se-GPX), selenium-binding protein (SeBP), heat shock protein 90 (HSP90) and ferritin (FT) which related to the oxidation/reduction system in abalone Haliotis discus hannai Ino were first cloned and analyzed under the conditions of nutritional regulation respectively.In addition, other relative genes (Cu/Zn-SOD, Mn-SOD, CAT, GST, Tpx, HSP70, HSP26 and NF-κB, etc.)were also detected in abalone fed with graded diets containing vitamin C or a-lipoic acid using real-time PCR assays, respectively.1 Molecular cloning, characterization and mRNA expression of selenium-dependent glutathione peroxidase from abalone Haliotis discus hannai Ino in response to dietary selenium, zinc and ironA novel selenium-dependent glutathione peroxidase (Se-GPX) was cloned from abalone Haliotis discus hannai Ino (HdhGPx) by homology cloning with degenerate primers and RACE techniques. The full length of HdhGPx cDNA was 963 bp with a 669 bp open reading frame (ORF) encoding 222 amino acids and a 101 bp eukaryotic selenocysteine insertion sequence (SECIS) in 3'untranslated region (UTR).It was showed that HdhGPx has a characteristic codon at 235TGA237 that corresponds to selenocysteine (SeC) as U72. Sequence characterization revealed that HdhGPx contains a characteristic GPx signature motif 2 (96LGLPCNQF103), an active site motif (179WNFEKF184). In Addition, two potential N-glycosylation sites (112NGTE115 and 132NLTQ135) were identified in HdhGPx.3D modeling analysis showed that the overall structure of HdhGPx monomer had more similarity to human GPx3.Relatively higher-level mRNA expression was detected in hepatopancreas, mantle and gonad by real-time PCR assays. The relative expression levels of HdhGPx mRNA in hepatopancreas and haemocytes were detected by real-time PCR in abalone fed with nine different diets containing graded levels of selenium (0.15,1.32 and 48.7 mg/kg), zinc (6.69,33.85 and 710.63 mg/kg) and iron (29.17,65.7 and 1267.2 mg/kg) for 20 weeks, respectively. The results showed that the expression of HdhGPx mRNA increased and reached the maximum at optimal dietary selenium(1.32 mg/kg), zinc (33.85 mg/kg) and iron (65.7 mg/kg), respectively.But HdhGPx mRNA expression levels were down-regulated by excessive dietary selenium (48.7 mg/kg), zinc (710.63 mg/kg) and iron(1267.2 mg/kg) and zinc (33.85 mg/kg) compared with these dietary minerals, respectively. These results indicated that optimal dietary minerals could trigger the mRNA expression of HdhGPx to increase the total antioxidant capacities in abalone.2 Molecular cloning, characterization and mRNA expression of selenium-binding protein in abalone (Haliotis discus hannai Ino):response to dietary selenium, iron and zincSelenium-binding protein(SEBP) is believed to play crucial role in controlling the oxidation/reduction in the physiological processes. In this study, the cDNA of selenium-binding protein from abalone Haliotis discus hannai Ino (HdhSEBP) was cloned by homology cloning and rapid amplification of cDNA ends (RACE) technique.The full length of HdhSEBP cDNA was 2071 bp, consisting of a 5'untranslated region (UTR) of 55 bp,a 3'UTR of 522 bp, and an open reading frame (ORF) of 1494 bp. The deduced protein has 497 amino acid residues with a calculated molecular mass of 55.6 kDa and a predicted isoelectric point of 5.47.BLAST analysis reveals that HdhSEBP shares high identities with other known SEBPs from mammal, bird, fish and mollusk, etc.The mRNA expression patterns of HdhSEBP in hepatopancreas and haemocytes were measured by real-time PCR in abalone fed with nine different diets containing graded levels of selenium (0.15,1.32 and 48.7 mg/kg), iron (29.17,65.7 and 1267.2 mg/kg) and zinc (6.69, 33.85 and 710.63 mg/kg) for 20 weeks, respectively. The results showed that the expression of the HdhSEBP mRNA increased and reached the maximum at optimal dietary selenium(1.32 mg/kg), iron (65.7 mg/kg) and zinc (33.5 mg/kg), respectively. Deficient or excessive level of dietary selenium, iron or zinc, respectively, leaded to significant depression of HdhSEBP mRNA. It is concluded that the expression levels of HdhSEBP are probably involved in the regulation of oxidation/reduction homeostasis affected by dietary selenium, iron or zinc.3 Molecular Cloning, Characterization and expression analysis of heat shock protein 90 from abalone, Haliotis discus hannai Ino in response to dietary selenium,iron and zincIn the present study, the cDNA of Haliotis discus hannai Ino HSP90 (designated HdhHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of HdhHSP90 was of 2660 bp, including an open reading frame (ORF) of 2187bp encoding a polypeptide of 728 amino acids with predicted molecular weight of 84.134kDa and theoretical isoelectric point of 4.619.BLAST analysis revealed that HdhHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in HdhHSP90, which indicated that HdhHSP90 should be a cytosolic member of the HSP90 family.Relatively higher levels of HdhHSP90 mRNA expression were detected in gonad and haemocytes by real-time PCR assays. The expression levels of HdhHSP90 in haemocytes and hepatopancreas were measured by real-time PCR after abalones were fed with different semipurified diets containing selenium (0.15,1.32 and 48.7 mg/kg), zinc (6.69,33.85 and 710.63 mg/kg) and iron (29.17,65.7 and 1267.2 mg/kg) supplements for 20 weeks, respectively. The results showed that the expression of the HdhHSP90 mRNA increased and reached the maximum at optimal dietary selenium(1.32 mg/kg), iron (65.7 mg/kg) and zinc (33.5 mg/kg), respectively.Deficient or excessive level of dietary selenium, iron or zinc, respectively, leaded to significant depression of HdhHSP90 mRNA.It is concluded that the expression of HdhHSP90 could be affected by dietary minerals and were involved in the anti-oxidation stress functions arose by dietary selenium, iron or zinc. 4 Transcriptional up-regulation of a novel ferritin homolog in abalone Haliotis discus hannai Ino by dietay ironA novel cDNA encoding ferritin (HdhNFT) was cloned from the hepatopancreas of abalone, Haliotis discus hannai Ino. The deduced protein contains 171 amino acid residues with a predicted molecular weight (MW) about 19.8 KDa and theoretical isoelectric point (pI) of 4.792.Amino acid alignment revealed that HdhNFT shared high similarity with other known ferritins. The HdhNFT contained a highly conserved motif for the ferroxidase center, which consists of seven residues of a typical vertebrate heavy-chain ferritin with a typical stem-loop structure. HdhNFT mRNA contains a 27 bp iron-responsive element (IRE) in the 5'-untranslated region. This IRE exhibited 82.14% similarity with abalone H. discus discus and 78.57% similarity with Pacific oyster Crassostrea gigas IREs. By real-time PCR assays, the mRNA transcripts of HdhNFT were found to be higher expressed in kidney, hepatopancreas, gill, mantle and muscle than in haemocytes and gonad. Moreover, mRNA expression levels of HdhNFT in the hepatopancreas and haemocytes were measured by real-time PCR in abalone fed with graded levels of dietary iron (29.2,65.7,1267.2 and 6264.7 mg/kg). Results showed that the expression of the HdhNFT mRNA increased with dietary iron contents. Furthermore, the maximum value of the HdhNFT mRNA was found in the treatment with 6264.7 mg/kg of dietary iron. These data indicated that dietary iron can up-regulate HdhNFT at transcriptional level in abalone Haliotis discus hannai Ino.5 Effect of vitamin C supplements on antioxidant defence and stress proteins in hepatopancreas of abalone Haliotis discus hannai InoStudies were conducted to investigate the effects of dietary ascorbic acid on transcriptional expression of antioxidant proteins, stress responsive proteins and nuclear factor Rel/NF-κB in hepatopancreas of abalone Haliotis discus hannai Ino (initial average length:84.36±0.24 mm) using real-time quantitative PCR assays. Four practical diets were formulated to contain 0.0,70.3,829.8 and 4967.5 mg ascorbic acid equivalent kg-1 diet, supplied as L-ascorbyl-2-polyphosphate (LAPP). Each diet was fed to triplicate groups of abalone adult in seawater floating acrylic tank (200 L), and each cage was stocked with 15 abalone adult. Abalone were fed once daily(17:00) to apparent satiation for 24 weeks. The results showed that adequate vitamin C (70.3 mg/kg) could significantly up-regulate expression levels of Cu/Zn-SOD, CAT,GST, NFT and HSP26 in hepatopancreas of abalone fed with dietary vitamin C supplement compared with abalone fed with vitamin C-free diet. But the expression levels of CAT, GST and HSP26 were decreased in abalone fed with excessive dietary vitamin C (70.3 mg/kg) compared with abalone fed adequate dietary vitamin C (70.3 mg/kg).And adequate vitamin C (70.3 mg/kg) could significantly down-regulate expression levels of Mn-SOD, GPX,TPx,SEBP, HSP70, HSP90 in hepatopancreas of abalone compared with abalone fed with vitamin C-free diet. In addition, significant up-regulations of expression levels of Mn-SOD, GPX,TPx, SEBP, NFT, HSP70, HSP90 and Rel/NF-κB were observed in abalone fed with excessive dietary vitamin C (829.8 and 4967.5 mg/kg) compared with abalone fed adequate dietary vitamin C (70.3 mg/kg). It is concluded that adequate dietary vitamin C could affect the expression levels of antioxidant proteins,stress responsive proteins and nuclear factor Rel/NF-KB in hepatopancreas of abalone at transcriptional level. Meanwhile, adequate dietary vitamin C could reduce the oxidative stress partly though the coordinated effect with antioxidant proteins in abalone.In conclusion, adequate dietary minerals (selenium, zinc and iron) or antioxidant (vitamin C) could affect the mRNA expression of antioxidant gene, increase the total antioxidant capacities of abalone H. discus hannai, and then eliminate or reduce the oxidative stresses caused by kinds of ROS in abalone H. discus hannai. |
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