(Solen grandis) ,, SgFer S (Ferritin) 4500 (Recalcati et al, 2008),

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1 44 3 Vol.44, No OCEANOLOGIA ET LIMNOLOGIA SINICA May, 2013 (Solen grandis) * 1, , 2 1, ( ; ; ; ) (Solen grandis) (SgFer) cdna, 848bp, bp 212bp, 525bp, 174, 20.2kDa, 5.20 PCR SgFer,, SgFer, SgFer (PGN) (glucan), mrna, 6h 12h ; (LPS) mrna SgFer ; ; ; PCR S968.3 (Ferritin),, 24, 163, 4500 (Recalcati et al, 2008),,, :, (IRE), ;, IRE, (Orino et al, 2001; Petit et al, 2001; Jyoti et al, 2007),,, (, 2009),,, (Fenneropenaeus chinensis) (Fenneropenaeus indicus), (WSSV), (Mohankumar et al, 2006; Zhang et al, 2006) (Penaeus Vanmamei) (, 2011), (Eriocheir sinensis) (Kong et al, 2010),,, *, ;, ;,, yangdinglong12@163.com :, lxq6808@163.com : , :

2 3 : (Solen grandis) 665 (Crassostrea gigas) (Gueguen et al, 2003), (Asterias forbesi) mrna (LPS) (Beck et al, 2002), (Solen grandis), cdna cdna,, PCR, (LPS) (PGN) (glucan), PCR,,, (Solen grandis), 85mm, SgFer cdna cdna, 2038 EST, EST BLAST, EST, Trans 5α ( ),, pbluescript II SK* M13F M13R, SgFer 1.3 SgFer SgFer : BLAST ; SingalP ; Clustal W ( ; MEGA 4.1, (Neighbor-joining), 1000 bootstraps 1.4 PCR SgFer 5,,, Trizol (Invitrogen) RNA, DNA (Promega) DNA, M-MLV (Promega) cdna, 80 Mastercycler ep realplex (Eppendorf) PCR PCR 25μL, cdna μL, 12.5μL 2 SYBR Green Master Mix (Applied Biosystems), 1μL 8μL SgFer SgFer-F: 5 -TTCA- GCCCATTCAGAAGCCA-3, SgFer-R: 5 -AGGTTGG- TGATGCGGTCGTT-3, β-actin : β-actin AF: 5 -TGTACGCCAACACTGTCCTG- TC-3, β-actin AR: 5 -CATCGTATTCCTGCTTGCTG- ATC min; 95 5s, 60 31s, 40 2 ΔΔCT (Livak et al, 2001), t, P<0.05, P< SgFer PBS (LPS) (PGN) (glucan), mg/ml 200, 40, 50μL, 1 3 LPS PGN glucan, 4 PBS, 5 (Yang et al, 2011) h 5,, 4 800g 5min,, 1ml TRIzol, RNA, cdna, PCR (Yang et al, 2011) 2 ΔΔCT, t, P<0.05, P< SgFer cdna SgFer cdna 848bp (GenBank : JX462607), 5 3 (UTR) 111bp 212bp, (ORF)525bp, 174 SignalP, 5 UTR CAGTGA, (IRE) 3 UTR Poly(A) AATAAA ExPASy pi 5.20, 20.2kDa; N112 N- N-Q-S-L, E28 Y33 E62 E63 H66 E108 Q142 ( 1)

3 ; SgFer,, ( 3) 2.4 SgFer PCR SgFer, : SgFer,, ;, ; ( 4) 2.5 SgFer PCR LPS PGN glucan SgFer, : PGN, SgFer 6h, 2.80 (P<0.01); 12h, 24h 1.57 ; 48h 0.40 (P< 0.05) SgFer glucan 12h (P<0.01), 4.80,, 48h 0.63 LPS, SgFer ( 5) 3 1 SgFer Fig.1 Nudeotide and deduced amino acid sequences of SgFer : ; N- ; ; IRE ; ; SgFer SgFer, 80% 73% 73%, 9 5, 7 ( 2) 2.3 SgFer SgFer 14, MEGA 4.1, : 14 3 Heavy(H) Light(L) H, L, L, H (Theil, 2003; Kim et al, 2004), H 7 SgFer, 7 (Zhang et al, 2003; Yang et al, 2011),, SgFer 7 : Glu28 Tyr33 Glu62 Glu63 His66 Glu108 Gln142, Glu63 His66 Fe, Tyr35 Tyr30 Tyr33, (Beck et al, 2002) 3 5, 5 (Zak et al, 2003; Zhang et al, 2007) SgFer cdna 5 (IRE), (IRP)

4 3 : (Solen grandis) SgFer Fig.2 Alignment of the amino acids sequence of SgFer with ferritins from other species : SgFer, : (CAD91440), (AEN83774), (AAX55641), (ADX31290), (AFG26289), (AAB20316), (NP_034369), (AAA35832) A E: 5 α ; : 7 ; :, IRP/IRE, ;, IRP/IRE, (Proudhon et al, 1996),,,, ;, (Linder et al, 1992; Huang et al, 1996) SgFer 3 SgFer Fig.3 Phylogenetic tree based on sequences of SgFer with ferritins from other species : MEGA 4.1, GenBank : (AAA35832), (NP_034369), (ADI24353), (AAB20316), (AAX61132), (AFG26289), (AEN83774), (AAZ20754), (AAX55641), (ADD17345), (XP ), (EAT40926), (AFH06683)

5 Fig.4 4 SgFer mrna SgFer mrna expression level in different tissues,, SgFer PGN glucan, 6h 12h, (Gueguen et al, 2003) SgFer PGN glucan, PGN glucan,, SgFer SgFer PGN glucan,,, LPS SgFer, LPS, SgFer,, 5 SgFer mrna Fig.5 SgFer mrna expression level after microorganism glycan stimulation, SgFer, (, 2010;, 2010; Huang et al, 1996; Zhang et al, 2003; Hsieh et al, 2006),,, (Zhang et al, 2003) SgFer,, (, 2009;, 2010) (LPS) (PGN) (glucan),,, , 34(1): 51 56,, , 30(4): 42 47,,, , 29(6): 7 10,,, , 34(6): Beck G, Ellis T W, Habicht G S et al, Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule. Developmental and Comparative Immunology, 26(1): Gueguen Y, Cadoret J P, Flament D et al, Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas. Gene, 303: Huang T S, Law J H, Soderhall K, Purification and cdna cloning of ferritin from the hepatopancreas of the freshwater crayfish Pacifastacus leniusculus. European Journal of Biochemistry, 236(2): Jyoti V, Margaret C, Anthony R et al, The role of ferritins in the physiology of Salmonella enterica. Typhi-murium: a unique role for ferritin B in iron-sulphur cluster repair and virulence. Molecular Microbiology, 63(5):

6 3 : (Solen grandis) 669 Kim S R, Lee K S, Yoon H J et al, Molecular cloning, expression and characterization of cdnas encoding the ferritin subunits from the beetle, Apriona germari. Comparative Biochemistry and Physiology, Part B: Biochemistry & Molecular Biology, 138: Kong P F, Wang L L, Zhang H et al, Two novel secreted ferritins involved in immune defense of Chinese mitten crab Eriocheir sinensis. Fish & Shellfish Immunology, 2: 1 9 Linder M C, Madani N, Middleton R et al, Ferritin synthesis on polyribosomes attached to the endoplasmic reticulum. Inorg Biochem, 47(3 4): Livak K J, Schmittgen T D, Analysis of relative gene expression data using real-time quantitative PCR and the 2 (T) (Delta Delta C) method. Methods, 25: Mohankumar K, Ramasamy P, White spot syndrome virus infection decreases the activity of antioxidant enzymes in Fenneropenaeus indicus. Virus Research, 15(1): Orino K, Lehman L, Tsuji Y et al, Ferritin and the response to oxidative stress. Biochemical Engineering Journal, 357(1): Petit J M, Briat J F, Lobréaux S, Structure and differential expression of the four members of the Arabidopsis thanliana ferritin gene family. Biochemical Engineering Journal, 359(3): Proudhon D, Wei J, Briat J F et al, Ferritin gene organization: different between plants and animals suggest possible kingdom-specific selective constraints. Journal of Molecular Evolution, 42(3): Recalcati S, Invernizzi P, Arosio P et al, New functions for an iron storage protein: the role of ferritin in immunity and autoimmunity. Journal of Autoimmunity, 30: Theil E C, Ferritin: at the crossroads of iron and oxygen metabolism. Journal of Nutrition, 133: 1549S 1553S Yang J, Wang L, Zhang H et al, C-type lectin in Chlamys farreri (CfLec-1) mediating immune recognition and opsonization. PLoS One, 6:e17089 Zak O, Aisen P, Iron release from transferrin, its C-Lobe, and their complexes with transferrin receptor: presence of N-Lobe accelerates release from C-Lobe at endosomal ph. Biochemistry, 42(42): Zhang J Q, Li F H, Wang Z Z et al, Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis. Journal of Biotechnology, 125: Zhang X J, Qu G, Zhu W L et al, Construction of intestinal cdna library and analysis of some expressed sequence tags sequencing of Ctenopharyngodon idellus. Acta Hydrobiologica Sinica, 31(2): Zhang Y, Meng Q X, Jiang T M et al, A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata). Comparative Biochemistry and Physiology. Part B: Biochemistry and Molecular Biology, 135(3): CLONING OF A FERRITIN GENE FROM SOLEN GRANDIS AND ITS RESPONSE TO MICROORGANISM GLYCAN ON TRANSCRIPTION LEVEL YANG Ding-Long 1, 2, WEI Xiu-Mei 2, YANG Jian-Min 2, YANG Jia-Long 3, XU Jie 1, 2, WANG Sheng 1, 2, LIU Xiang-Quan 2, ZHANG Yan-Min 4 (1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, ; 2. Shandong Provincial Key Laboratory of Restoration for Marine Ecology, Shandong Marine Fishers Research Institute, Yantai, ; 3. Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, ; 4. Shandong Business Institute, Yantai, ) Abstract In this study, the full length cdna of a ferritin gene from Solen grandis (designed as SgFer) was cloned, and it was 848bp and consisted of a 5 untranslated region (UTR) of 111bp, a 3 UTR of 212bp and an open reading frame (ORF) of 525bp encoding a polypeptide of 174 amino acids with an estimated molecular mass of 20.2kDa and isoelectric point of The tissue and temporal expression of SgFer after microorganism glycan challenge was recorded by real-time PCR. SgFer transcript could be detected in all examined tissues with the highest expression level in hepatopancreas. The mrna expression of SgFer was up-regulated after PGN or glucan stimulation, and reached peak at 6h and 12h post-challenge, respectively. No significant changes were observed after LPS stimulation. SgFer might be involved in elimination of microorganism glycan. Key words Solen grandis; Ferritin; Gene cloning; Real-time PCR