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1 , 20(6): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 王鹤, 胡宝庆, 文春根, 胡向萍, 程周玉, 宁瑞红,, 摘要 : (Cristaria plicata) ( ) cm ( ) cm, 1 40, 20 Trizol h RNA, cdna cdna, 514 bp, 5 UTR 57 bp; 3 UTR 242 bp, poly(a) ; 192 bp, 63, N 1 23, 40, 8.72, 7.13 kd α β, CSαβ 10 9 cell/ml (Aeromonas hydrophila), PCR,,, 1.25, h,, ; 12 h,, 0 h, ; 24 h 36 h,,, : ; ; ; ; ; 中图分类号 : Q 文献标志码 : A 文章编号 : (2013) (mytilin),, [1] Boman (Enterobacter cloacae) (Escherichia coli) (Hyatophora cecropins) Cecropins [2] 2 000, [1, 3],,,, [3] ; α- β- θ- 5 [4] ; [5] 1980 Lehrer (Lepus hainanus) 收稿日期 : ; 修订日期 : 基金项目 : ( ; ); (2013FB09); (GJJ12024; GJJ10378); (2004). 作者简介 : (1989-),,, @qq.com 通信作者 :,,,. cgwen@ncu.edu.cn

2 6 : 1189, defensin, α-,, [1] (Suncus murinus) (Homo sapiens) (Cavia porcellus) (Macaca arctoides), (paneth cell) α- [5] β- (Capra ibex), (Mus musculus) (Rattus norvegicus), (Bos gaurus) [6] θ- (Macaca mulatta) [7] NP-1 [8] (Gallus gallus domesticus) (Sus scrofa)β- [9 10], Defb48 [11], (Ruditapes philippinarum) [12] (Crassostrea gigas) [13] (Mytilus edulis) [14 16] (Haliotis discus) [17] (Musca domestica) [18] (Epinephelus drumondhayi) [19] (Hyriopsis cumingii) [20] (Cristaria plicata), (Aeromonas hydrophila),,,, , ( ) cm ( ) cm, 1, (25 1), 1 ( XS91-4-1), - 2~3 d, 28~30, UV-3100, Gel Doc XR, PCR (K640 ), (5417R), (VD-650 ), (MCO-15AC ), (DYY-10C JY600PJ ) EDTA Typetone , cell/ml 0.1 ml, PBS RNA Trizol h RNA, 1% RNA RNA cdna, PCR (GenBank: KC290726) β-actin cdna, ( 1) PCR 25 μl H 2 O 18.4 μl, 10 PCR buffer 2.5 μl, dntps 2 μl, defensinf 0.6 μl, defensinr 0.6 μl, extaq 0.3 μl 94 5 min, s, s, 72 1 min, 35, min β-actin PCR 94 5 min, s, s, 72 1min, 35 ; min 表 1 用于 PCR 扩增的褶纹冠蚌防御素和内参 β-actin 基因的特异引物 Tab. 1 The special primers of defensin and β-actin for PCR amplification in Cristaria plicata primer β-actinf β-actinr defension BF defension BR (5' 3')sequence(5' 3') CTTGACTTGGCAGGTAGAGA CAGACAGCACAGTGTTAGCA GCATATGCATTTGCACTCC ATGTCCAGTTGGGGATGTC

3 PCR 1%, PCR mrna cdna cdna, ExPASY( pku.edu.com) ( ) ( ) BLAST ( nih.gov/blast/) SignalP 3.0( SignalP/) Clustal W1.83 ; CLUSTAL X 1.83 MEGA 4 ; MotifScan SMART ; 2 25 GenBank Quantity One β-actin, SPSS cdna cdna 514 bp, 5 UTR 57 bp; 3 UTR 242 bp, poly(a) ; (Open Reading Frame, ORF) 192 bp, 63 ( 1) SignalP 3.0, N 1 23 MRVAVVLALVLLLAVIDIPQAAA, , 7.13 kd, 8, Ser27, 2 7, MotifScan SMART 表 2 Tab. 2 species Ruditapes philippinarum Hirudo medicinalis Theromyzon tessulatum Aplysia californica Hydra magnipapillata Hyriopsis cumingi Haliotis discus Ornithodoros moubata Crassostrea gigas Mytilus edulis Polistes dominulus Anopheles sinensis Culex quinquefasciatus Aedes aegypti Anomala cuprea Hyblaea puera Acalolepta luxuriosa Notostigma carazzii Rossomyrmex minuchae Cataglyphis velox Lasius japonicus Musca domestica Stomoxys calcitrans Drosophila melanogaster Cristaria plicata 25 种动物 cdna 序列登录号 GenBank numbers of 25 species GenBank GenBank Accession number AFP50008 ABV56569 AAR12065 ABF21076 ABE26989 ACQ90304 FJ BAB41028 ACS91537 P81610 ADB85559 AAY21793 XP AAD40115 BAD77967 ABQ08056 AAK35160 ACB46515 ACB46514 ACB46513 ACB46519 ABM66377 AAD56536 AAF58855 KC Defensin-2 Arthropod defensin α β, α β ( 2) 2.2, 1, 1, (Ornithodoros moubata),,

4 6 : ( ) ;. Fig.1 Nucleotide and deduced amino acid sequences of defensin cdna from Cristaria plicata The letters in boxes(asterisk)represented iniation codon(atg)and termination codon(taa); Underline showed Signal peptide sequence. 2 A. [13], B. α., B.. Fig. 2 Three dimensional structure comparison of defensin protein from Cristaria plicata and Crassostrea gigas A. indicates Crassostrea gigas defensin [13] ; B. indicates Cristaria plicata defensin. α. indicates helix structure; β. indicates folding structure. (Hirudo medicinalis) (Theromyzon tessulatum) ( 3) 2.3,, 1.25, ( 4) 2.4 PBS, 120 bp β-actin 370 bp, PBS 3 25 NJ Fig. 3 Neighbor-joining phylogentic tree of defensin amino acid sequences based on 25 species animals, ( 5) 2.5, 12 h,, 48 h ( 6a);

1192 20 12 h, 24 h (P<0.05), ( 6b);, 12 24 h, 24 h,, 48 h, 0 h( 6c); (P<0.05), PBS, 48 h PBS 4 (P>0.05, n=3, x ± SD) Fig.

5 h, 24 h (P<0.05), ( 6b);, h, 24 h,, 48 h, 0 h( 6c); (P<0.05), PBS, 48 h PBS 4 (P>0.05, n=3, x ± SD) Fig. 4 Expression of defensin gene in different tissues from Cristaria plicata No significant difference among different tissues (P>0.05, n=3, x ± SD) 5 PBS H. ; G. ; L. ; M.. Fig. 5 Expression of defensin in different tissues from Cristaria plicata by injecting PBS H. haemocytes; G. gill; L. hepatopancreas; M. mantle. ( 6 d) PBS, (P>0.05) PBS,,,, 48 h, ;, PBS, 48 h 3 Fig. 6 6 * (P 0. 05, n=3, x ± SD). Expression of defensin in different tissues from Cristaria plicata by injection Aeromonas hydrophila * showing significant difference(p 0. 05, n=3, x ± SD) with control.

6 6 : 1193 α- β-, CSαβ, α- β-,, [21] cdna 201 bp, 66, 44, N 22, α β, 8 4 (C 1 ~C 4, C 2 ~C 5, C 3 ~C 7, C 6 ~C 8 ) [12] Cg-def cdna 323 bp, 195 bp, 65, N 22, CSαβ 8 4 [13] MGD1 MGD2, 21 39, CSαβ 8 4 [15] cdna 414 bp, 73, 8 kd, 37 N, CSαβ 6 3 (C 1 ~C 3, C 2 ~C 5, C 4 ~C 6 ) [22] cdna 514 bp, 192 bp, 63, 23 ;, CSαβ, 8 4 ;, 1.5,, [14],,, ( : AAR12065) 97, 22, α β, 10 5 : C 1 ~C 3 C 2 ~C 6 C 4 ~C 5 ; C 7 ~C 8 C 9 ~C 10, 113, [20],, [23 24] (Staphylococcus aureus) COS-7 (human beta-defensin3, hbd3),, hbd3 [25] ML-35p (Pseudomonas aeruginosa)atcc27853,,, ( LPS) [26] β- 3(Gal-3) Gal-3, ; Gal-3,, Gal-3 [27], Mdde, Mdde ; Mdde, Mdde,, Mdde, [28] (HcBD),, 4 h, HcBD, 72 h, HcBD [20] (Vibrio anguillarum),,

7 ,,,, 24 h, [17],, (Microcococcus lysodeikticus) 24 h 48 h,, [13] MCdef,, (Streptococcus iniae) KCTC3651 (Staphylococcus aureus) KCTC1916, [12] (Ornithodoros tick), (Enterococcus faecalis) (methicillin- resistant Staphylococcusaureus), [29] (Haemaphysalis longicornis) Hlgut Hlsal, G1 G2 S1 S2, G1 S1, G2 S2 C α β, G1 S1, α β G2 S2 ; Hlgut Hlsal α β [30],,,,, [22] (Micrococcus luteus), 7, 24 h MGD1, 48 h ; 24 h, 48 h ; ; mytilin B MGD2 [14 16],,, 12 h ; 12 h ; 24 h,, 参考文献 : [1],,,. [J]., 2007, 27(1): [2],,,. [J]., 2010, 9(4): [3],. [J]., 2009, 1(4): [4],,. [J]., 2003, 10(2): [5] Risso A. Leukocyte antimicrobial peptides: multifunctional effector molecules of innate immunity[j]. J Leukoc Biol, 2000, 68(6): [6],,. β- cdna [J]., 2004, 26(3): [7],,,. [J]., 2008, 4(3): 1 3. [8],,,. NP-1 [J]., 2000, 27(11): [9],,,. β- [J]., 2008, 39(8): [10],. β- (pbd) [C]., 2009(4):

8 6 : 1195 [11],,,. Defb48 [J]., 2010, 6(4): [12] Adhya M, Jeung HD, Kang HS, et al. Cloning and localization of MCdef, a defensin from Manila clams(ruditapes philippinarum )[J]. CBP, 2012, 12(161): [13] Gueguen Y, Herpin A, Aumelas A, et al. Characterization of a defensin from the oyster Crassostrea gigas[j]. J Biol Chem, 2006, 281(1): [14] Charlet M, Chernysh S, Philippe H, et a1. Innate immunity isolation of severa1 cysteinerich antimicrobia1 peptides from the blood of a mollusk, Mytilus edulis[j] J Biol Chem, 1996, 271(36): [15] Mitta G, Vandenbulcke F, Hubert F, et al. Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge[j]. J Cell Science, 1999, 12(112): [16] [16] Mitta G, Hubert F, Dyrynda EA, et al. Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis[j]. Dev Comp Immunol, 2000, 4(24): [17],,,. hd-def cdna [J]., 2008, 18(3): [18],,,. cdna [J]., 2003, 49(3): [19],,. β- [J]., 2011, 35(5): [20],,,. Big defensin cdna [C]//2011, [21],,,. [J]., 4(1): [22] Nakajima Y, Taylor D, Yamakawa M, et al. Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata(acari: Argasidae)[J]. Insect Biochem Molec, 2001, 8(31): [23] Otvos LJr. Antibacterial peptides isolated from insects[j]. J Pept Sci, 2000, 10(6): [24] Reddy KVR, Yedery RD, Aranha C. Antimicrobial peptides: premises and promises[j]. Int J Antimicr Agents, 2004, 24(6): [25],,,. β- 3 COS-7 [J]., 2004, 28(4): [26],,,. β- 2 [J]., 2002, 18(1): [27],,,. β 3 [J]., 2006, 28(4): [28],,,. [J]., 2010, 53(6): [29] Nakajima Y, Ishibashi J, Yukuhiro F. Antibacterial activity and mechanism of action of tick defensin against Gram-positive bacteria[j]. BBA, 2003, 1624(1): [30] Zhou JL, Liao M, Ueda M, et al. Sequence characterization and expression patterns of two defensin-like antimicrobial peptides from the tick Haemaphysalis longicornis[j]. Peptides, 2007, 5(28):

9 Gene characterization and expression of defensin in Cristaria plicata WANG He, HU Baoqing, WEN Chungen, HU Xiangping, CHEN Zhouyu, NING Ruihong Department of Bioscience, Institute of Life Science, Nanchang University, Nanchang , China Abstract: The specimen of Cristaria plicata with shell length( ) cm and shell height( ) cm were temporally cultured for one week. We designed test trial and control trial with 20 individuals for each. We obtained the full-length cdna sequence of defensin from the cdna library of Cristaria plicata. The full-length cdna was 514 bp, consisting of a 5'-terminal untranslated region(utr)of 57 bp, a 3'-terminal UTR of 242 bp with a poly(a)tail, and an open reading frame of 192 bp which encoded a polypeptide of 63 amino acids residues, including 23 signal peptides and 40 mature peptides. The predicted molecular weight was 7.13 kd, the theoretical isoelectric point was The predicted structure revealed that defensin gene contained a conserved cysteine-stabilized α-helix and two β-sheet(csαβ)structural motif. We measured the expression defensin in the hemocytes, mantle, gill, and hepatopancreas of C. plicata by semi-quantitative RT-PCR following stimulation by Aeromonas hydrophila(10 9 cell/ml). Before stimulation, the expression of defensin was highest in the mantle and was about 1.25 times that in hemocytes and gill. After injection(at 0, 6, 12, 24, 36, 48 h), the relative expression first decreased in the mantle and hepatopancreas then increased significantly in the control group. There was no change in defensin expression in the hemocytes and gill. In the experimental group, expression peaked after 12 h in the hemocytes then decreased. Conversely, defensin expression was highest in the other tissues at 0 h, and then decreased gradually. Expression increased slightly in the mantle and gill at 24 h and 36 h. Key words: Cristaria plicata; defensin; gene expression; sequence structure; mytilin; Aeromonas hydrophila Corresponding author: WEN Chungen. cgwen@ncu.edu.cn

2004级研究生现代免疫学实验技术结业报告

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