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1 , 20(6): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 李洋 1,2, 刘萍 2, 李健 2, 李吉涛 2, 马朋 2 2, 高保全 1., ; 2.,, 摘要 : RACE (Exopalaemon carinicauda) serpin cdna bp, bp, 414, kd, 5.694, (Penaeus monodon), 49%, serpin,, serpin 8 h (P<0.05), serpin 2 h 24 h, (P<0.05), serpin cdna,, : ; ; ; ; 中图分类号 : Q959 文献标志码 : A 文章编号 : (2013) (serine protease inhibitor, SPI),,, [1 3] [4 6],,,, [7 9], 74, Serpin Kazal pacifastin [8] (Manduca sexta) (Eriocheir sinensis) (Fenneropenaeus chinensis) (Penaeus monodon) [10 13] Serpins, Serpin [11 12, 14 15] serpin (Exopalaemon carinicauda), (Palaemonidae) (Palaemon) (Exopalaemon),, [16],, 收稿日期 : ; 修订日期 : 基金项目 : 863 (2012AA10A409); (CARS-47); ( ) ( ); (2013A0701). 作者简介 : (1984 ),,,. liyang_able@163.com 通信作者 :,. liuping@ysfri.ac.cn

2 6 : 1167, ( ph ),,,,,,,,,, [17], [18], cdna,, 1 1.1, (4.50±0.50) cm 200 L PVC 1, 21, 32, ph 8.0,, , 3, 135, h 15, 5, 800 g 10 min Trizol 1.2 RNA cdna,, 0.05 g 1 ml Trizol, Trizol RNA, RNA, 1.5%, RQ1 RNase-free DNase RNA DNA cdna (25 μl) : 1 μl(1μg/μl) RNA Oligo dt2.5 μl H 2 O 5 μl, 72 5 min, 80 2 min, 5 μl 5 buffer, 1.25 μl dntp(10 mmol/l), μl RNase inhibitor, 1.25 μl M-MLV, DEPC μl,, 42 1 h, min cdna NCBI GenBank (Portunus trituberculatus)(afa42363) (Marsupenaers japonicus)(bai50776) (ABC33916) (Scylla paramamosain) (ACY66635) serpin, DNAMAN, F1 R1 F2 R2( 1),, Blast primer Serpin F1 Serpin R1 Serpin F2 Serpin R2 Serpin F3 Serpin R3 Serpin F4 Serpin R4 18S HF 18S HR 表 1 实验中所用的引物序列 Tab. 1 Primer sequences used in this study (5 3 ) sequence of primer (5 3 ) AGMGGCGTGGYGCRTGGC ACGGCGAGRYGYTKGTTCA TTCAACTACVARKMYGAGGA GGCTCCMACKCGCGTRTA CCCAACAAGGCTACTGCCAGAACTAAAG CACTGGTAGAGATGCGTGAACTGGATGC AGGCGCGAATCTACTCTCCA CCACTCATCACCAAAGACATCAA TATACGCTAGTGGAGCTGGAA GGGGAGGTAGTGACGAAAAAT cdna, PCR, DNA, pmd18-t,

1168 20 Top10, PCR ( ) 1.4 serpin 3 RACE 5 RACE serpin 3 5 RACE ( 1), SMART TM RACE 3 5 RACE,, 1.5 NCBI VecScreen (http: //www.ncbi. nlm.nih.gov/vecscreen/vecscreen.html), NCBI BLAST (http: //blast.

3 Top10, PCR ( ) 1.4 serpin 3 RACE 5 RACE serpin 3 5 RACE ( 1), SMART TM RACE 3 5 RACE,, 1.5 NCBI VecScreen (http: // nlm.nih.gov/vecscreen/vecscreen.html), NCBI BLAST (http: //blast.ncbi.nlm.nih.gov/blast/), DNAMAN, EditSeq, SMART Interpro Scan, ClustalX MEGA 1.6 serpin RNA, cdna, serpin cdna, Serpin F3 Serpin R3( 1), RNA 18S HF 18S HR( 1), RT-PCR RT-PCR (20 μl) : SYBR Premix Ex Taq TM Ⅱ(2 ), 10 μl; PCR Forward Primer(10 μmol/l), 0.8 μl; PCR Reverse Primer(10 μmol/l), 0.8 μl; ROX Reference Dye Ⅱ(50 )*3, 0.4 μl; DNA, 0.6 μl; ddh 2 O, 7.4 μl : s; 95 5 s, s, 40 ; s, 60 1 min, s 3, RNA cdna, RT-PCR serpin PCR ± ( x ±SD), ΔΔ C T 2 serpin, SPSS serpin cdna cdna 700 bp serpin cdna, NCBI, (ADC42877), 49% RACE Serpin F3 Serpin R3 3 5 RACE, bp 500 bp ( 1) 1 serpin cdna M: DNA ; A serpin cdna ; B serpin cdna 5 RACE ; C serpin cdna 3 RACE. Fig. 1 The amplification results of serpin gene cdna sequence from E. carinicauda M: DNA ladder; A, cdna fragment of serpin gene; B, 5 RACE result of serpin gene; C, 3 RACE result of serpin gene bp serpin, (ORF) bp, 414, bp 88 bp, polya ( 2), kd, serpin cdna GenBank( : JQ838045) SMART, serpin serpin (46~406) 3 SignalP 4.0, 4, serpin 1~19

4 6 : serpin cdna ATG TAA,,. Fig. 2 cdna sequence and deduced amino acid sequence of serpin gene from E. carinicauda The letters in box indicated the start codon (ATG) and the stop codon (TAA). The signal pepetide was shaded and two binding sites for glycosylation were double underlined. 2.2 BlastP, serpin (ADC42877) (AFA42363) (ACY66635) (Branchiostoma lanceolatum)(cad68157)

1170 20 3 serpin Fig. 3 Signal peptide and functional domain of serpin from E. carinicauda 4 serpin Fig.

0 serpin 6, serpin,,, 2.3 serpin RT-PCR serpin,, serpin,, 2.

5 serpin Fig. 3 Signal peptide and functional domain of serpin from E. carinicauda 4 serpin Fig.4 The signal peptide result of serpin sequence of E. carinicauda 49% 43% 44% 32% MEGA 4.0 serpin 6, serpin,,, 2.3 serpin RT-PCR serpin,, serpin,, 2.4 serpin ( 8), serpin, 8 h, serpin 11> 46> 4> 39> 18> Fig. 5 5 serpin serpins Amino-acid sequence alignment of E. carinicauda serpin with different animals serpins

6 : 1171 6 MEGA4 NJ serpin Fig. 6 NJ tree based on serpin amino acid sequences using MEGA 4.0 (P<0.05)( 9),, 8 h, (P<0.05),, 24 h,, 48 h, (P<0.05) Fig.

6 6 : MEGA4 NJ serpin Fig. 6 NJ tree based on serpin amino acid sequences using MEGA 4.0 (P<0.05)( 9),, 8 h, (P<0.05),, 24 h,, 48 h, (P<0.05) Fig. 7 7 serpin Real time-pcr analysis of serpin gene expression from different tissues of E. carinicauda 25> 32( ), 32 h, 4 11, 18 39, (P<0.05) serpin 2 4 h 3 350~400, 8~9 α 3 β- β-, C, P1 [6, 19] serpin cdna, bp, serpin Fig.8 The expression of serpin in haemocyte after salinity stress of E.carinicauda

7 serpin Fig.9 The expression of serpin in hepatopancreas of E. caxinicauda after salinity stress, kd SMART, 19, 46~406 serpin, serpin [10] serpin, serpin [15], serpin serpin, serpin,, serpin serpin, serpin, serpin serpin, serpin, [11, 13] ; serpin, [20] serpin, serpin,,, serpin,, serpin PO SP, [21] [22 25] 7, PCR serpin, 32 h serpin ; serpin 4 h, 8 h, 24 h, serpin serpin, serpin, [11] ; serpin,, [15] ;, mrna, [26] serpin,, 参考文献 : [1] Ashida M, Sasaki T. A target protease activity of serpins in insect hemolymph[j]. Insect Biochem Molec, 1994, 24: [2] Simser JA, Palmer AT, Munderloh UG, et al. Isolation of a spotted fever group rickettsia, Richettsia peacockii, in a Rocky Mountain wood tick, Dermacentor andersoni, cell line[j]. Appl Environ Microb, 2001, 67:

8 6 : 1173 [3] Sritunyalucksana K, Soderhall K. The propo and clotting system in crustaceans[j]. Aquaculture, 2000, 191: [4] Ye S, Goldsmith EJ. Serpins and other covalent protease inhibitors[j]. Curr Opin in Struc Biol, 2001, 11: [5] Gettings PG. Serpin structure, mechanism and function[j]. Chem Rev, 2002, 102: [6] Van Gent D, Sharp P, Morgan K, et al. Serpins: structure, function and molecular evolution[j]. Int J Biochem Cell B, 2003, 35: [7] Jiang H, Wang Y, Gu Y, et al. Molecular identification of a bevy of serine proteinases in Manduca sexta hemolymph[j]. Insect Biochem Molec, 2005, 35: [8] Zdobnov E M, Von Mering C, Letunic I, et al. Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster[j]. Science, 2002, 298: [9] Simonet G, Claeys I, Franssens V, et al. Genomics, evolution and biological functions of the pacifastin peptide family: a conserved serine protease inhibitor family in arthropods[j]. Peptides, 2003, 24: [10] Jiang H, Wang Y, Yu X Q, et al. Prophenoloxidaseactivating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1j and serine proteinase homologs[j]. Insect Biochem Molec, 2003, 33: [11] Liu Y C, Li F H, Wang B, et al. A serpin from Chinese shrimp Fenneropenaeus chinensis is responsive to bacteria and WSSV challenge[j]. Fish Shellfish Immunol, 2009, 26(3): [12]. EST [D]. :, [13] Homvises T, Tassanakajon A, Somboonwiwat K. Penaeus monodon SERPIN, PmSERPIN6, is implicated in the shrimp innate immunity[j]. Fish Shellfish Immunol, 2010, 29(5): [14]. [D]. :, [15]. [D]. :, [16]. [M]. :, 1955: [17] Brito R, Chimal ME, Rosas C. Effect of salinity in survival, growth and osmotic capacity of early juveniles of Farfante Penaeus brasiliensis (DeeaPoda: Penaeidae)[J]. J Exp Mar Biol Ecol, 2000, 244: [18]. [D]. :, [19] Rawlings ND, Barrett AJ, Bateman A. MEROPS: the peptidase database[j]. Nucleic Acids Res, 2010, 38: [20] Liang Z, Soderhall K. Isolation of cdna encoding a novel serpin of crayfish hemocytes[j]. Comp Biochem Physiol B Biochem Mol Biol, 1995, 112: [21] Cerenius L, Soderhall K. The prophenoloxidase activating system in invertebrates[j]. Immunol Rev, 2004, 198: [22] Cheng W, Chen JC. Effects of environmental factors on the immune responses of freshwater prawn Macrobranchium rosenbergii and other decapod crustaceans[j]. Fishs Soc, 2002, 29: [23] Le Moullac G, Haffner P. Environmental factors affecting immune responses in Crustacea[J]. Aquaculture, 2000, 191: [24] Perazzolo LM, Gargioni R, Ogliari P, et al. Evaluation of some hemato-immunological parameters in the shrimp Farfantepenaeus paulensis submitted to environmental and physiological stress[j]. Aquaculture, 2002, 214: [25] Daniel B, Kim S, David R. The effect of low salinity on phenoloxidase activity in the Sydney rock oyster, Saccostrea glomerata[j]. Aquaculture, 2006, 251: [26] Zou Z, Jiang H. Manduca sexta serpin-6 regulates immune serine proteinases PAP-3 and HP8. cdna cloning, protein expression, inhibition kinetics, and function elucidation[j]. J Biol Chem, 2005, 280:

9 Cloning and expression analysis of serine protease inhibitors gene of Exopalaemon carinicauda LI Yang 1, 2, LIU Ping 2, LI Jian 2, LI Jitao 2, MA Peng 1, GAO Baoquan 2 1. Ocean University of Dalian, Dalian , China; 2. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China Abstract: Exopalaemon carinicauda belongs to Palaemonidae, Palaemon, and Exopalaemon, which is an important demersal species in the Yellow Sea and Bohai Sea. Its production ranks only second to that of Fenneropenaeus chinensiss and Acetes chinensis in China. This species is mainly charactered by its tender meat, high reproduction, fast growth and adaption to a wide range of environment. Recently, the aquaculture area has been expanded gradually, but under the condition of centralization-breeding factor, the shrimp is easy in stressing conditions, which may cause diseases even death. In this study, we isolated the cdna sequence of serine protease inhibitors (serpin) gene from Exopalaemon carinicauda using RT-PCR and RACE methods. The full-length cdna of the serine protease consisted of bp with a bp open reading frame (ORF), encoding 414 amino acids. The predicted molecular mass of serine protease protein was kd, and the theoretical isoelectric point was The serine protease protein sequence contained two glycosyl binding sites, which had 49% homology with that of Penaeus monodon. Tissue expression analysis suggested that serpin mrna expression was highest in the hemocytes and lowest in the muscle. Real time-pcr analysis revealed that the expression of serpin gene was significantly higher in hemolymph 8 h after salinity exposure compared to the control group. Furthermore, the serpin gene expression in the hepatopancreas exhibited two obvious peaks at 2 h and 24 h after salinity stress compared with the control group. Our results suggest that serpin gene plays an important role in the salinity stress response of E. carinicauda. Key words: Exopalaemon carinicauda; tissue expression; cloning; salinity stress; stress response Corresponding author: LIU Ping. liuping@ysfri.ac.cn

2004级研究生现代免疫学实验技术结业报告

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