6 : LITAF 737 Abstract: The lipopolysaccharide-induced TNF-alpha factor (LITAF) is an inflammatory cytokine, which plays an important role in innate i

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1 HEREDITAS (Beijing) , 34(6): ISSN 研究报告 DOI: /SP.J 虾夷扇贝脂多糖诱导的肿瘤坏死因子 LITAF 基因的克隆及表达分析赫崇波 1,2, 朱宝 1, 刘卫东 2, 鲍相渤 2, 李云峰 2, 单忠国 3, 李宏俊 1., ; 2.,, ; 3., ; 4., 脂多糖诱导的肿瘤坏死因子 (Lipopolysaccharide-induced TNF-alpha factor, LITAF) 是一类重要的炎症细胞因子, 在先天性免疫系统中发挥重要的介质作用文章根据虾夷扇贝 LITAF 基因 EST 序列, 应用 RACE 技术克隆了虾夷扇贝 LITAF 全长 cdna, 对序列及编码的氨基酸进行生物信息学分析结果显示, 该基因 cdna 全长 bp, 其 5 非编码区包含 76 bp, 3 非编码区包含 bp; 开放阅读框 (ORF) 为 474 bp, 编码 157 个氨基酸, 氨基酸序列中存在一个保守的 LITAF 结构域 ; 理论分子量 kda, 等电点为 6.24 LITAF 基因序列为 bp, 由 3 个外显子和两个内含子组成利用实时荧光定量 PCR 技术分析 LITAF 在虾夷扇贝不同组织不同胚胎发育阶段以及鳗弧菌 (Vibrio anguillarum) 刺激后各时间段的表达情况结果表明 :LITAF 基因在所检测的 6 个成体组织中均有表达, 其中肾脏的表达量最高 ; 胚胎发育的 7 个时期中, 担轮幼体时期表达量最高 ; 菌刺激 36 h 实验组与对照组的表达量差异大 LITAF 基因是 LITAF 家族的一员, 推测 LITAF 基因参与虾夷扇贝的先天性免疫反应虾夷扇贝 ; 脂多糖诱导的肿瘤坏死因子 ; 序列分析 ; 胚胎发育 ; 基因表达 Cloning and expression analysis of lipopolysaccharide-induced TNF-α(LITAF) of Japanese scallop (Mizuhopecten yessoensis) HE Chong-Bo 1,2, ZHU Bao 1, LIU Wei-Dong 2, BAO Xiang-Bo 2, LI Yun-Feng 2, SHAN Zhong-Guo 3, LI Hong-Jun 4 1. College of Life Science, Liaoning Normal University, Dalian , China; 2. Key Lab of Marine Fishery Molecular Biology of Liaoning Province, Liaoning Ocean and Fisheries Science Research Institute, Dalian , China 3. College of Oceanography and Environmental Science, Xiamen University, Dalian , China; 4. National Marine Environmental Monitoring Center, Dalian , China 收稿日期 : ; 修回日期 : 基金项目 : ( ) ( ) 作者简介 :,,, hechongbo@hotmail.com 网络出版时间 : :44:34 URL:

2 6 : LITAF 737 Abstract: The lipopolysaccharide-induced TNF-alpha factor (LITAF) is an inflammatory cytokine, which plays an important role in innate immunity system. Based on the expressed sequence tag (EST) of Japanese scallop (Mizuhopecten yessoensis), the cdna of LITAF gene was amplified using rapid amplification of cdna ends (RACE) approach. Results showed that the full-length cdna of LITAF is bp consisting of a 5 untranslated region (UTR) of 76 bp, a 3' UTR of bp, and an open reading frame (ORF) of 474 bp encoding a polypeptide of 157 amino acids, and there is a conserved LITAF domain in amino acid sequences. The estimated molecular mass is kda and the theoretical isoelectric point is The total length of LITAF is bp, which includes three exons and two introns. Real-time quantitative PCR was carried out to measure LITAF mrna expression in adult tissues and monitor mrna expression patterns during embryonic development after bacteria (Vibrio anguillarum) challenged. The expression level of LITAF mrna was detected in all the adult tissues with the highest in the kidneys. The trochophore owns the highest expression level of LITAF in embryonic development. LITAF expression showed significant difference(p<0.01)between the control and bacteria challenged specimens at 36 h. These results suggest that the LITAF should be a member of the LITAF family that perhaps involved in the innate immune response of Japanese scallop. Keywords: Japanese scallop(mizuhopecten yessoensis); lipopolysaccharide-induced TNF-alpha factor(litaf); sequence analysis; embryonic development; gene expression,, [1] (Lipopolysaccharide, LPS) α (Tumor necrosis factor alpha, TNF-α) TNF-α [2~5], [6], [7,8] α, TNF-a, (Lipopolysaccharide-induced TNF-alpha factor, LITAF) [9] [10~13], LITAF LPS TNF-α, LITAF STAT6(B), TNF-α [14] (Mizuhopecten yessoensis), [15] 1982,, 20, [16],,, cdna EST, PCR cdna, LITAF, LITAF LITAF,, 1 1.1, D, (Vibrio anguillarum)

3 738 HEREDITAS (Beijing) 总 RNA 的提取和 cdna 的获得 30 mg, RNAprep pure Tissue Kit( ) RNA, RNA Reverse Transcriptase M-MLV (TaKaRa ), min, 85 5 s 虾夷扇贝 LITAF 基因的克隆 cdna LITAF EST (GenBank GR867954), FastPCR 3 Race LITAF-F1 LITAF-F2( 1), RNA, 3 Race 3 RACE cdna, 3 Race PCR PCR pmd19-t (TaKaRa), DH5α, PCR 94 3 min; s, s, 72 2 min, 35 ; min LITAF mrna ( 1), DNA LITAF, 5, 3 PCR 94 1 min; s, min, 35 ; min PCR, 统计分析 ( ac.jp/form2.html), SignalP 3.0 Server ( services/signalp/), NCBI BLAST SPSS 13.0, ± (one way ANOVA), Student t-test P<0.05, P< 实时荧光定量 PCR 6 RNA D 7 RNA, cdna PCR,, CFU/mL 0.5 ml, 0.5 ml, h, RNA cdna 3 RACE cdna QLITAF-F QLITAF-R, β-actin ( 1) SYBR Premix Ex Taq (Takara), Mx 3005P PCR PCR 15 μl, 2 SYBR Premix Ex Taq 7.5 μl, ROX II 0.1 μl, 0.3 μl, cdna 1.5 μl, 5.3 μl s; s, s, s, 40 3, 2 ΔΔCt, LITAF 表 1 LITAF 基因序列扩增和实时荧光定量 PCR 所用的引物 (5 3 ) LITAF-F1 GCAAACAATCTGGACCAGCAGTG cdna 3 Race LITAF-F2 TTCAGTGATGACACATTGTGGAG LITAF-F3 CACCAATCTTTGTCAGTGCTCTAG LITAF-R1 CCTAATCCCTCAAGGAATGAAGC QLITAF-F ATCAGCACAGCCCTGGAATACG PCR QLITAF-R CACTTGTGCTGGACATCAGACAG Qβ-actin F AGTCCCAATCTACGAAGGTTATG Qβ-actinR CCAGTGATGAGGAGGAAGCAG

4 6 : LITAF LITAF cdna LITAF EST, 3 RACE PCR cdna (GenBank JF708046), bp, 474 bp, bp, bp, 4 mrna (ATTTA) (AATAAA), polya LITAF bp, 3, bp 245 bp, - GT-AG 2.2, kda, 6.24,, 65.2%, 13%, LITAF,, (Haliotis diversicolor supertexta)litaf 57%, (Danio rerio) (Mus musculus) (Homo sapiens)litaf 49% 47% 45%, (Chlamys farreri) (Meretrix meretrix)litaf 44% 2.3 LITAF LITAF 基因在 6 种组织的表达情况 PCR LITAF mrna 6, LITAF mrna 6 β-actin,, LITAF, ( 3) LITAF 基因在不同发育时期的表达 PCR LITAF D 7, LITAF 7,, D, ( 4) 图 3 LITAF 在虾夷扇贝各组织中的相对表达情况 a b c d, (P>0.05), (P<0.05) 图 4 LITAF 在虾夷扇贝不同发育时期的相对表达情况 a b c,, (P<0.05) LITAF 基因在鳗弧菌刺激后的表达 PCR ( 5),, LITAF,, 12 h 36 h, 36 h, 2 (P<0.01) 3,

5 740 HEREDITAS (Beijing) LITAF,, [1,21,22], [19,23], LITAF, LITAF 参考文献 (References): [1] Loker ES, Adema CM, Zhang SM, Zhang SM, Kepler TB. 图 5 鳗弧菌刺激后不同时间虾夷扇贝血 LITAF 的相对表达情况 * P<0.05; ** P<0.01, LITAF, STAT6(B) TNF-a [14], LITAF [10~13], LITAF LITAF N CXXC 25 C (H)XCXXC,, N C CXXC, Zn 2+ [17] LITAF, [14,18] LITAF,, LITAF, [11,19], LITAF,,,,, D [20], LITAF,,, Invertebrate immune systems - not homogeneous not simple, not well understood. Immunol Rev, 2004, 198(1): [2] Chappell VL, Le LX, LaGrone L, Mileski WJ. STAT proteins play a role in tumor necrosis factor alpha gene expression. Shock, 2000, 14(3): [3] Marrack P, Kappler J. T cells can distinguish between allogeneic major histocompatibility complex products on different cell types. Nature, 1988, 332(6167): [4],,. TNF-α., 2004, 26(6): [5] Bazzoni F, Beutler B. The tumor necrosis factor ligand and receptor families. N Engl J Med, 1996, 334(26): [6] García-Castillo J, Pelegrín P, Mulero V, Meseguer J. Molecular cloning and expression analysis of tumor necrosis factor α from a marine fish reveal its constitutive expression and ubiquitous nature. Immunogenetics, 2002, 54(3): [7] Pfeffer K. Biological functions of tumor necrosis factor cytokines and their receptors. Cytokine Growth Factor Rev, 2003, 14(3-4): [8] Hehlgans T, Pfeffer K. The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily: players, rules and the games. Immunology, 2005, 115(1): [9] Tang XR, Fenton MJ, Amar S. Identification and functional characterization of a novel binding site on TNF-α promoter. Proc Natl Acad Sci USA, 2003, 100(7): [10] Hong YH, Lillehoj HS, Lee SH, Park DW, Lillehoj EP. Molecular cloning and characterization of chicken lipopolysaccharide-induced TNF-α factor (LITAF). Dev Comp Immunol, 2006, 30(10): [11] Yu YD, Qiu LM, Song LS, Zhao JM, Ni DJ, Zhang Y. Molecular cloning and characterization of a putative

6 6 : LITAF 741 lipopolysaccharide-induced TNF-alpha factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri. Fish Shellfish Immunol, 2006, 23(2): [12] Park EM, Kim YO, Nam BH, Kong HJ, Kim WJ, Lee SJ, Kong IS, Choi TJ. Cloning, characterization and expression analysis of the gene for a putative lipopolysaccharide-induced TNF-alpha factor of the Pacific oyster, Crassostrea gigas. Fish Shellfish Immunol, 2008, 24(1): [13] Zhang DC, Jiang JJ, Jiang SG, Ma JJ, Su TF, Qiu LH, Zhu CY, Xu HP. Molecular characterization and expression analysis of a putative LPS-induced TNF-alpha factor (LITAF) from pearl oyster Pinctada fucata. Fish Shellfish Immunol, 2009, 27(3): [14] Tang XR, Marciano DL, Leeman SE, Amar S. LPS induces the interaction of a transcription factor, LPS-induced TNF alpha factor, and STAT6(B) with effects on multiple cytokines. Proc Natl Acad Sci USA, 2005, 102(14): [15] Nagashima K, Sato M, Kawamata K, Nakamura A, Ohta T. Genetic structure of Japanese scallop population in Hokkaido, analyzed by mitochondrial haplotype distribution. Mar Biotechno, 2005, 7(1): [16],,,,,.., 2009, 31(6): [17] Ponting CP, Mott R, Bork P, Copley RR. Novel protein domains and repeats in Drosophila melanogaster: insights into structure, function, and evolution. Genome Res, 2001, 11(12): [18] Bolcato-Bellemin AL, Mattei MG, Fenton M, Amar S. Molecular cloning and characterization of mouse LITAF cdna: role in the regulation of tumor necrosis factor-alpha (TNF-alpha) gene expression. J Endotoxin Res, 2004, 10(1): [19] Li HJ, Yang Q, Gao XG, Su H, Wang J, He CB. Identification and expression of a putative LPS-induced TNF-α factor from Asiatic hard clam Meretrix meretrix. Mol Biol Rep, 2012, 39(2): [20] Culliney JL. Larval development of the giant scallop Placopecten magellanicus (Gmelin). Biol Bull, 1974, 147(2): [21] Lacoste A, De Cian MC, Cueff A, Poulet SA. Noradrenaline and alpha-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells. J Cell Sci, 2001, 114(19): [22] Lacoste A, Cueff A, Poulet SA. P35-sensitive caspases, MAP kinases and Rho modulate β-adrenergic induction of apoptosis in mollusc immune cells. J Cell Sci, 2002, 115(4): [23] Shan ZG, Li HJ, Bao XB, He CB, Yu HN, Liu WD, Hou L, Wang J, Zhu D, Sui LJ, Zhu B, Li YF. A seleniumdependent glutathione peroxidase in the Japanese scallop, Mizuhopecten yessoensis: cdna cloning, promoter sequence analysis and mrna expression. Comp Biochem Physiol B Biochem Mol Biol, 2011, 159(1): 1 9.

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附件3 广东省高水平大学重点学科建设项目申报书项目名称 : 基于南海现代渔业可持续发展的水产学科建设申报单位 : 广东海洋大学 ( 盖章 ) 主要依托学科 : 所属领域 : 水产畜牧学机械工程信息与通信工程农学项目负责人 : 谭北平联系电