首页 >  水产学报 >  魁蚶过氧化氢酶基因克隆及表达分析

2016, 40(6): 856-866. doi: 10.11964/jfc.20151010106

魁蚶过氧化氢酶基因克隆及表达分析

1. 中国水产科学研究院黄海水产研究所, 农业部海洋渔业可持续发展重点实验室, 山东 青岛 266071;

2. 上海海洋大学水产与生命学院, 上海 201306

通讯作者: 刘志鸿, liuzh@ysfri.ac.cn

收稿日期:2015-10-12
修回日期:2016-03-09

基金项目:   科技基础性工作专项(2013FY110700)  黄海所基本科研业务费项目(2060302201516054)  科技基础条件平台项目(2060503-D1)  山东省自然科学基金(ZR2013CQ047) 

关键词: 魁蚶 , 过氧化氢酶 , RACE , qRT-PCR

Gene cloning and expression analysis of catalase in Scapharca broughtonii

1. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;

2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

Corresponding author: LIU Zhihong, liuzh@ysfri.ac.cn

Received Date:2015-10-12
Accepted Date:2016-03-09

Keywords: Scapharca broughtonii , catalase , RACE , qRT-PCR

采用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆出魁蚶过氧化氢酶(SbCAT)基因cDNA全长序列,该基因全长为2181 bp,包括1431 bp的开放阅读框(open reading frame,ORF),96 bp的5'端非翻译区(UTR)和654 bp的3'-UTR。其中ORF编码477个氨基酸,预测分子量为54 ku,理论等电点为8.03。SbCAT氨基酸序列与其他所选动物的氨基酸序列具有较高的相似性,其相似度为68%~96%,SbCAT氨基酸具有CAT基因家族的特征性序列,包括CAT活性位点,1个亚铁血红素结合位点及3个催化位点残基。此外,SbCAT还具有保守的亚铁血红素结合口袋与还原型辅酶Ⅱ(NADPH)结合位点。采用实时荧光定量PCR(qRT-PCR)检测了SbCAT的组织表达特征。结果显示,SbCAT mRNA在所检测的6种组织中均有表达,在外套膜中表达量较高,在肝胰腺和红细胞中表达量较低。经鳗弧菌和金黄色葡萄球菌刺激后,鳗弧菌刺激组的外套膜表达量一直较低,其他组织均表现出先上升再下降的趋势。研究表明,SbCAT可能在魁蚶免疫防御中发挥重要作用。

Catalase(EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to water and oxygen. In the present study, the complete cDNA of Catalase in ark shell Scapharca broughtonii(named SbCAT) was cloned by RT-PCR and rapid amplification of cDNA ends technique, which contained 2181 bp. The cDNA consists of a 5' untranslated region(UTR) of 96 nucleotides, the 3' UTR of 654 bp, and an open reading frame(ORF) of 1431 bp, encoding 477 amino acid residues with 54 ku predicted molecular weight and the theoretical isoelectric point of 8.03. SbCAT amino acid sequence compared with the other animals compared 68%-96%.The deduced amino acid sequence of SbCAT has characteristic features of catalase family such as the catalase active site(61FNRERIPERVV-HAKGAG77), the catalase heme-ligand signature motif(351RLFSYPDTH359) and the three catalytic amino acid residues of His-72, Asn-145 and Tyr-355. In addition, SbCAT also has the conservative heme-binding pocket and NADPH binding sites. Quantitative real-time PCR(qRT-PCR) method was used to analyze the SbCAT mRNA expression characterization in tissues of normal ark shells. The results showed that SbCAT mRNA detected was expressed in six had a high similarity of and it was higher in the mantle and lower in the tissues of hepatopancreas and haemocyte. After Vibrio anguillarum and Staphylococcus aureus challenge, SbCAT expression was rather low in mantle with V.anguillarum challenged, and significantly up-regulated in other tissues. This results suggests that SbCAT may play an important role in the immune defense of S. broughtonii.

参考文献

[1] 王颖, 吴志宏, 李红艳, 等. 青岛魁蚶软体部营养成分分析及评价[J]. 渔业科学进展, 2013, 34(1):133-139. Wang Y, Wu Z H, Li H Y, et al. Analysis and evaluation of nutrition composition in soft tissue of Anadara uropygimelana[J]. Progress in Fishery Sciences, 2013, 34(1):133-139(in Chinese).
[2] 陈竞春, 石安静. 贝类免疫生物学研究概况[J]. 水生生物学报, 1996, 20(1):74-78. Chen J C, Shi A J. Malacozoan immunobiology research:a review[J]. Acta Hydrobiologica Sinica, 1996, 20(1):74-78(in Chinese).
[3] 周丽青, 杨爱国, 王清印, 等. 魁蚶血细胞分类及其免疫功能的初步分析[J]. 水产学报, 2013, 37(4):599-606. Zhou L Q,Yang A G,Wang Q Y,et al. Studies on the hemocytes types and their immunological functions in bloody clam(Scapharca broughtonii)[J]. Journal of Fisheries of China, 2013, 37(4):599-606(in Chinese).
[4] Zheng L B, Wu B, Liu Z H, et al. A manganese superoxide dismutase(MnSOD) from ark shell, Scapharca broughtonii:Molecular characterization, expression and immune activity analysis[J]. Fish & Shellfish Immunology, 2015, 45(2):656-665. DOI:10.1016/j.fsi.2015.05. 003.
[5] Zheng L B, Liu Z H, Wu B, et al. Ferritin has an important immune function in the ark shell Scapharca broughtonii[J]. Developmental & Comparative Immunology, 2016,59:15-24. DOI:10.1016/j.dci.2015.12.010.
[6] 郑利兵, 吴彪, 刘志鸿, 等. 魁蚶(Scapharca broughtonii)半乳糖凝集素(SbGal)基因cDNA的克隆及表达分析[J]. 海洋与湖沼, 2015, 46(5):1061-1070. Zheng L B, Wu B, Liu Z H, et al. Cloning and expression analysis of galectin from Scapharca broughtonii(SbGal)[J]. Oceanologia Et Limnologia Sinica, 2015, 46(5):1061-1070(in Chinese).
[7] Li M, Zhu L, Zhou C Y, et al. Molecular characterization and expression of a novel big defensin(Sb-BDef1) from ark shell, Scapharca broughtonii[J]. Fish & Shellfish Immunology, 2012, 33(5):1167-1173. DOI:10.1016/j.fsi.2012.09.008.
[8] Kashiwagi A, Kashiwagi K, Takase M, et al. Comparison of catalase in diploid and haploid Rana rugosa using heat and chemical inactivation techniques.[J]. Comparative Biochemistry & Physiology-Part B:Biochemistry and Molecular Biology, 1997, 118(3):499-503. DOI:10.1016/S0305-0491(97)00216-2.
[9] Nishikawa M, Hashida M, Takakura Y. Catalase delivery for inhibiting ROS-mediated tissue injury and tumor metastasis[J]. Advanced Drug Delivery Reviews, 2009, 61(4):319-326. DOI:10.1016/j.addr.2009.01.001.
[10] 李建喜, 杨志强, 王学智. 活性氧自由基在动物机体内的生物学作用[J]. 动物医学进展, 2006, 27(10):33-36. Li J X, Yang Z Q, Wang X Z. Biological Function of Reactive Oxygen Free Radicals in Animals[J]. Progress in Veterinary Medicine, 2006, 27(10):33-36(in Chinese).
[11] Dröge W. Free Radicals in the Physiological Control of Cell Function[J]. Physiological Reviews, 2002, 82(1):47-95. DOI:10.1152/physrev.00018.2001.
[12] Suzuki Y J, Forman H J, Sevanian A. Oxidants as Stimulators of Signal Transduction[J]. Free Radical Biology and Medicine, 1997, 22(1-2):269-285. DOI:10.1016/S0891-5849(96)00275-4.
[13] Quan F, Korneluk R G, Tropak M B, et al. Isolation and characterization of the human catalase gene[J]. Nucleic Acids Research, 1986, 14(13):5321-5335. DOI:10.1093/nar/14.13. 5321.
[14] Reimer D L, Bailley J, Singh S M. Complete cDNA and 5' genomic sequences and multilevel regulation of the mouse catalase gene.[J]. Genomics, 1994, 21(2):325-336. DOI:10.1006/geno.1994.1273.
[15] Hideaki N, Mikio Y, Kiminobu G, et al. Isolation and characterization of the rat catalase-encoding gene[J]. Gene, 1989, 79(2):279-288. DOI:10.1016/0378-1119(89)90210-2.
[16] Zhang Y, Fu D K, Yu F, et al. Two catalase homologs are involved in host protection against bacterial infection and oxidative stress in Crassostrea hongkongensis[J]. Fish & Shellfish Immunology, 2011, 31(6):894-903. DOI:10.1016/j.fsi.2011.08.005.
[17] Guo H Y, Zhang D C, Cui S, et al. Molecular characterization and mRNA expression of catalase from pearl oyster Pinctada fucata[J]. Marine Genomics, 2011, 4(4):245-251. DOI:10.1016/j.margen. 2011.05.003.
[18] Li C H, Ni D J, Song L S, et al. Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri[J]. Fish & Shellfish Immunology, 2008, 24(1):26-34. DOI:10.1016/j.fsi.2007.06.010
[19] Ekanayake P M, Zoysa M D, Kang H S, et al. Cloning, characterization and tissue expression of disk abalone(Haliotis discus discus) catalase[J]. Fish & Shellfish Immunology, 2008, 24(3):267-278. DOI:10.1016/j.fsi.2007.11.007.
[20] 董迎辉. 泥蚶高通量转录组分析及生长相关基因的克隆与表达研究[D]. 青岛:中国海洋大学, 2012. Dong Y H. Transcriptome analysis using 454 pyrosequencing and cloning and expression of growth-related genes for the blood clam Tegillarca granosa(Linnaeus, 1758)[D]. QingDao:Ocean University of China, 2012(in Chinese).
[21] Klotz M G, Klassen G R, Loewen P C. Phylogenetic relationships among prokaryotic and eukaryotic catalases.[J]. Molecular Biology & Evolution, 1997, 14(9):951-958. DOI:10.1093/oxfordjournals. molbev.a025838.
[22] Putnam C D, Arvai A S, Bourne Y, et al. Active and inhibited human catalase structures:ligand and NADPH binding and catalytic mechanism[J]. Journal of Molecular Biology, 2000, 296(1):295-309. DOI:10.1006/jmbi.1999.3458.
[23] 高杉, 周遵春, 董颖, 等. 仿刺参过氧化氢酶基因全长cDNA的克隆及表达分析[J]. 中国农业科技导报, 2014, 16(2):127-134. Gao S, Zhou Z C, Dong Y, et al. Full-length cDNA cloning and expression analysis of catalase gene from sea cucumber(Apostichopus japonicus)[J]. Review of China Agricultural Science and Technology, 2014, 16(2):127-134(in Chinese).
[24] 王家红, 童玥, 朱玥, 等. 蛋白质糖基化的研究进展[J]. 药物生物技术, 2011, 18(1):77-80. Wang J H, Tong Y, Zhu Y, et al. The research progress in protein glycosylation[J]. Pharmaceutical Biote-chnology, 2011, 18(1):77-80(in Chinese).
[25] Amery L, Brees C, Baes M, et al. C-terminal tripeptide Ser-Asn-Leu(SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal.[J]. Biochemical Journal, 1998, 336(2):367-371. DOI:10.1042/bj 3360367.
[26] Rachubinski R A, Subramani S. How proteins penetrate peroxisomes[J]. Cell, 1995, 83(83):525-528. DOI:10. 1016/0092-8674(95)90091-8.
[27] Yang X L, Li G, Wen C G, et al. A catalase from the freshwater mussel Cristaria plicata with cloning, identification and protein characterization[J]. Fish & Shellfish Immunology, 2011, 31(3):389-399. DOI:10.1016/j.fsi.2011.06.003.
[28] Khessiba A, Roméo M, Aïssa P. Effects of some environmental parameters on catalase activity measured in the mussel(Mytilus galloprovincialis) exposed to lindane[J]. Environmental Pollution, 2005, 133(2):275-281. DOI:10.1016/j.envpol.2004.05.035
[29] 任虹, 李强, 李婷. 重金属污染物对文蛤金属酶类的影响[J]. 中国渔业质量与标准, 2014, 4(4):7-12. Ren H, Li Q, Li T. Effects of heavy metals on metalloenzymes from Meretrix meretrix[J]. Chinese Fishery Quality and Standards, 2014, 4(4):7-12(in Chinese).
[30] Levine A, Tenhaken R, Dixon R, et al. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response[J]. Cell, 1994, 79(4):583-593. DOI:10.1016/0092-8674(94)90544-4.
[31] Lambert C, Soudant P, Choquet G, et al. Measurement of Crassostrea gigas hemocyte oxidative metabolism by flow cytometry and the inhibiting capacity of pathogenic vibrios[J]. Fish & Shellfish Immunology, 2003, 15(3):225-240. DOI:10.1016/S1050-4648(02)00160-2.
[32] Liu C H, Tseng M C, Cheng W. Identification and cloning of the antioxidant enzyme, glutathione peroxidase, of white shrimp, Litopenaeus vannamei, and its expression following Vibrio alginolyticus infection[J]. Fish & Shellfish Immunology, 2007, 23(1):34-45. DOI:10.1016/j.fsi.2006.09.002.
[33] Abele-Oeschger D, Sartoris F J, Pörtner H O. Hydrogen peroxide causes a decrease in aerobic metabolic rate and in intracellular ph in the shrimp Crangon crangon[J]. Comparative Biochemistry and Physiology-Part C:Pharmacology, Toxicology and Endocrinology, 1997, 117(2):123-129. DOI:10.1016/S0742-8413(97)00059-5.
[34] Liu H P, Chen F Y, Gopalakrishnan S, et al. Antioxidant enzymes from the crab Scylla paramamosain:Gene cloning and gene/protein expression profiles against LPS challenge[J]. Fish & Shellfish Immunology, 2010, 28(5-6):862-871. DOI:10.1016/j.fsi.2010.02.008.
[35] Zhang Q L, Li F H, Zhang X J, et al. cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology, 2008, 24(5):584-591. DOI:10.1016/j.fsi.2008.01.008.
[36] 周丽青, 杨爱国, 王清印, 等. 鳗弧菌对魁蚶血细胞形态及免疫功能的影响[J]. 海洋与湖沼, 2014, 45(3):536-541. Zhou L Q, Yang A G, Wang Q Y, et al. Effect of Vibrio anguillarum on morphology and immunological function of blood cells in Scapharca broughtonii[J]. Oceanologia et Limnologia Sinica, 2014, 45(3):536-541(in Chinese).

相关文章

[1] 陈晶, 聂青, 刘妍. 《WHO基本药物示范目录》与我国《国家基本药物目录》动态调整程序比较与借鉴.水产学报,2015(3): 289-293.doi:10.3866/PKU.WHXB201503022
  • 导出引用
  • 下载XML
  • 收藏文章
计量
  • 文章下载量()
  • 文章访问量()

目录

魁蚶过氧化氢酶基因克隆及表达分析