(Vibrio anguillarum) (Cyclina sinensis) *

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1 43 4 Vol.43, No OCEANOLOGIA ET LIMNOLOGIA SINICA July, 2012 (Vibrio anguillarum) (Cyclina sinensis) * ( ) (GSTs), SMART cdna, σ (CsGSTS) PCR CsGSTS, CsGSTS cdna 793bp, 206, GST-N GST-C GSTs 6 24h (P<0.01), GSTs 6 12h (P<0.05), 48 96h (P<0.01); CsGSTS 3 6h, 24h, 48h, 3, (GSTs),, CsGSTS, Q789 (Cyclina sinensis),, (, 2010) (Vibrio anguillarum),, (, 2007), A, (, 2003) (Glutathione S-transferases, GSTs) (, 2009), GSTs (, 2004; Masella et al, 2005), (GSH),, GSTs (Armstong, 1997; Ivarsson et al, 2003; Shimada, 2006), (, 2006) /, GSTs 10, α μ π σ θ ω β κ ζ (Sheehan et al, 2001; Allocati et al, 2009; Wan et al, 2009), GSTs cdna CsGSTS,, (Cyclina sinensis),,, 5 (Chlorella sp.),, (Vibrio anguillarum) 2216E 37 24h, *, 12JCZDJC22800, kaiyaluo@126.com :,,, panbaoping@yahoo.com.cn : , :

736 43, 1 10 8 cell/ml 78, 36 50μl/, 36, 6 (0h) 0h 3h 6h 12h

2 736 43, cell/ml 78, 36 50μl/, 36, 6 (0h) 0h 3h 6h 12h 24h 48h 96h 6, 50mg,, RNA r/min 10min, GSTs 1 : 9, r/min 15min,, 1% 5%, ( ) :, 37 1min, GSH 1μmol/L cdna, 1ml TRIZOL (Catalog number: ) RNA, mrna QIAGEN Oligotex mrna Kits, cdna Clontech SMART cdna Library Construction Kit, pbluescript II SK+, E. coli (DH5α) T7-F: 5 -TAATACGACTCACTATAGG-3, T3-R: 5 -AATTAA CCCTCACTAAAGG-3 contig singlest, ESTs BLAST CsGSTS sigma cdna GenBank BLASTX, (Open Reading Frame, ORF), SMART, Clustal W, MEGA4.1 (NJ) CsGSTS, RNA cdna cdna, β-actin, RT- PCT : actin-f: 5 -CACCACAACTGCCGAGAG-3, actin-r: 5 -CCGATAGTGATGACCTGACC-3 ; CsGSTS- F: 5 -TATCGGACCAAAGATGACGC-3 ; CsGSTS-R: 5 - AGTTCTGGGTAGGAGG AGAGTAG-3 : 95 30s, 95 5s, 58 30s, 72 30s, 40 2 ΔΔCT (Livak et al, 2001), SPSS, CsGSTS GSTs, GSTs ( 1A) (0h) GSTs 18.3U/ml, 6h GST (P<0.01), 12h 68.5U/ml, 3.4, 24h, (P<0.01), 48 96h 96h, GSTs ( 1B) GSTs 1 GSTs Fig.1 Changes in GSTs activity in serum and liver after injection of V. anguillarum : #, (P<0.05), ## (P<0.01); **, (P<0.01)

3 4 : (Vibrio anguillarum) (Cyclina sinensis) U/mg prot, 6h 521.8U/mg prot, (P<0.05), 12h 495.7U/mg prot 24h, 48h (P<0.01), 96h 850.1U/mg prot, CsGSTS SMART cdna cfu/ml, 96.4%, BLASTX ORF σ CsGSTS cdna 739bp, ORF 621bp, 206 ( 2), 23.47kDa, pi=7.66, GST-N GST-C, N β-α-βα-β-β-α, C 5 α CsGSTS GenBank JN CsGSTS, CsGSTS Ruditapes philippinarum σ (GenBank : ADI ), 58% 5 σ GST ( 3), σ GST N (G ) C (H ), G (Y) (S), H, σ GST 15 σ GST ( 4), bootstrap 1000, σ GST (Ruditapes philippinarum) (Solen grandis), (Enteroctopus dofleini) (Nototodarus sloanii), 2.4 CsGSTS 96h, CsGSTS ( 5) 3h, CsGSTS, (P<0.05); 6h, (P<0.01), CsGSTS, 24h (P<0.05), 48h, 3, 96h, (P<0.05) CsGSTS 0h (P>0.05) 3, GSTs (Brian et al, 2002; Cheung et al, 2002), (, 2003) 2 CsGSTS cdna Fig.2 The open reading frame of full length cdna of CsGSTS gene and analysis on the structural domain :, *, GST-N, GST-C

4 σ GST Fig.3 Multiple alignments of sigma like GST between C. sinensis and other species : *, :, G, H HdGSTS: Haliotis discus discus (ABO26603); CgGSTS: Crassostrea gigas (CAE11863); RpGSTS: Ruditapes philippinarum (ADI ); SgGSTS: Solen grandis (AEW ) GSTs, GSTs 6 24h GSTs,,,, GSTs GSTs 6 12h,,,,, GSTs,, GSTs (, 2006) Aliya (2003), GSTs, N, /, GSH, GSH σ GST G, GST H,,, σ GST σ GST,, σ GST σ GST (2010), (Aeromonas hydrophila), 3 12h GST ; 3 6h, CsGSTS (2003),, GSTs, CsGSTS 4 (NJ) 16 σ GST Fig.4 The plylogenetic tree constructed by the amino acid sequences of sigma like GST of 16 species using NJ (Neighbor Joining) method

5 4 : (Vibrio anguillarum) (Cyclina sinensis) CsGSTS Fig.5 The expression of CsGSTS gene at different times in liver of C. sinensis infected by V. anguillarum : # (P<0.05), ## (P<0.01); * (P<0.05), ** (P<0.01) ( CsGSTS ), mrna CsGSTS 24h, 48h, (, 2010) (, 2010),,,, GSTs CsGSTS,,,,, , 4: ,,, S-., 4(2): ,, , 27(4): ,,, S., 45(4): ,,, (Vibrio anguillarum) (Cyclina sinensis)., 41(2): ,,, , 27(8): 11 15,,, S., 32(12): 85 91,,, (Cyclina sinensis) (Vibrio anguillarum)., 41(6): ,,, S-., 15(4): Aliya S, Reddanna P, Thyagaraju K, Does glutathione S-transferase Pi (GST-Pi) a marker protein for cancer. Molecular and Cellular Biochemistry, 253: Allocati N, Federici L, Masulli M et al, Glutathione S-transferrases in bacteria. FEBS Letters, 276: Armstong R N, Structure, catalytic mechanism, and evolution of the glutathione transferases. Chemical Research in Toxicology, 10: 2 18 Brian N B, Singh B R, Induction of glutathione S-transferase in the Northern Quahog Mercenaria mercenaria after exposure to the polychlorinated biphenyl (PCB) mixture aroclor Journal of Protain Chemistry, 21(8): Cheung C C C, Zheng G J, Lam P K S et al, Relationships between tissue concentrations of chlorinated hydrocarbons (polychlorinated biphenyls and chlorinated pesticides) and antioxidative responses of marine mussels Perna viridis. Marine Pollution Bulletin, 45: Ivarsson Y, Mackey A J, Edalat M et al, Identification of residues in glutathione transferase capable of driving functional diversification in evolution. A novel approach to protein redesign. Journal of Biological Chemistry, 278: Livak K J, Schmittgen T D, Analysis of relative gene expression data using Real-time quantitative PCR and the 2 ΔΔCT methods. Methods, 25: Masella R, Benedetto R D, Vari R et al, Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathonerelated enzymes. Journal of Nutritional Biochemistry, 16: Sheehan D, Meade G, Foley V M et al, Structure, function and evolution of glutathione S-thansferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochemical Journal, 360(Pt1): 1 16 Shimada T, Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons. Drug Metabolism and Pharmacokinetics, 21: Wan Q, Whang I, Lee J S et al, Novel omega glutathione S-transferases in disk abalone: Characterization and protective roles against environmental stress. Comparative Biochemistry and Physiology, 150:

6 EFFECT OF VIBRIO ANGUILLARUM ON ACTIVITY AND GENE EXPRESSION OF GLUTATHIONE S-TRANSFERASES IN CYCLINA SINENSIS LUO Kai-Ya, LIU Xin-Xin, GE Duan-Yang, PAN Bao-Ping (College of Life Sciences, Tianjin Key Laboratory of Cyto-Genetical and Molecular Regulation, Tianjin Normal University, Tianjin, ) Abstract The activities of glutathione S-transferases (GSTs) in serum and liver were tested under Vibrio anguillarum stress. The sigma-like glutathione S-transferases of Cyclina sinensis (CsGSTS) were cloned with SMART-cDNA library and large scale EST sequencing method, then the expression of CsGSTS in liver were detected by the real-time quantitative PCR. Results showed the GSTs activities in serum were up-regulated at 6 24h (P<0.01) after infection, while in liver the activities declined at 6 12h and increased significantly at 48 96h. The full length cdna of CsGSTS consisted of 793bp, encoding 206 amino acids with the typical GST-N and GST-C domain. The CsGSTS expression declined at 3 6h (P<0.05), and increased significantly at 24h, then reached its highest level at 48h, which was about three times higher than control group. Results showed that Vibrio may cause tissue injury at the first stage of infection, then plenty of GSTs were produced and CsGSTS expressed intensively, both participating in the immune response of the clam. This investigation provided important experimental data for the study on immunologic mechanism in C. sinensis. Key words Cyclina sinensis, Glutathione S-transferases (GSTs), Enzyme activity, CsGSTS gene, Gene expression

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