收稿日期: 2008-09-03
修回日期: 2009-03-30
网络出版日期: 2011-10-10
基金资助
国家科技部863项目(2007AA09Z437); 国家自然科学基金项目(30670367); 广东省科技计划项目(2007B020701002,
2005B20301005); 广东省自然科学基金项目(031886); 教育部留学回国人员科研启动基金项目
Molecular cloning and sequence analysis of pancreatic trypsinogen and amylase from orange-spotted grouper (Epinephelus coioides)
Received date: 2008-09-03
Revised date: 2009-03-30
Online published: 2011-10-10
Supported by
国家科技部863项目(2007AA09Z437); 国家自然科学基金项目(30670367); 广东省科技计划项目(2007B020701002,
2005B20301005); 广东省自然科学基金项目(031886); 教育部留学回国人员科研启动基金项目
采用RT-PCR及RACE法从斜带石斑鱼Epinephelus coioides肝胰脏克隆得到胰蛋白酶原(trypsinogen, TRY)与淀粉酶(amylase, AMY)基因cDNA全序列。斜带石斑鱼肝胰脏TRY基因cDNA全长911 bp, 其中5’非翻译区(5’-UTR)为55bp, 3’-UTR为127bp, 开放阅读框(ORF)为729bp, 编码242个氨基酸, 包含所有丝氨酸蛋白酶中共有的高度保守的催化活性中心。序列一致性分析发现, 斜带石斑鱼与牙鲆Paralichthys olivaceus、金头鲷Sparus aurata、鳎Solea senegalensis、石鲽Pleuronectes bicoloratus的TRY序列相似性高达86.8%—89.7%, 与人Homo sapiens、小鼠Mus musculus、斑马鱼Danio rerio 的TRY相似性较低为59.9%—64.5%。斜带石斑鱼AMY基因cDNA全长1657bp, 其中5’-UTR为41bp, 3’-UTR为77 bp, ORF为1539bp, 编码512个氨基酸, 包含与哺乳动物α- AMY二级结构相似的8个α螺旋和8个β折叠。序列一致性分析发现, 斜带石斑鱼与澳洲肺鱼Neoceratodus forsteri、美洲拟鲽Limanda americanus、大西洋鲑Salmo salar、斑马鱼AMY基因序列相似性高达82.4%—91.8%, 与人、小鼠、鸡G. Gallus的AMY基因相似性较低为70.1%—72.3%。斜带石斑鱼TRY和AMY基因cDNA全序列的成功克隆为进一步研究其表达调控机理及研发有效提高其表达水平的饲料添加剂奠定基础。
关键词： 胰蛋白酶原; 淀粉酶; 基因克隆; 序列分析; 斜带石斑鱼Epinephelus coioides
胡永乐,梁旭方,王琳,李观贵,刘秀霞,王云新,张海发 . 斜带石斑鱼胰蛋白酶原和淀粉酶全长cDNA的克隆与序列分析[J]. 热带海洋学报, 2010 , 29(5) : 125 -131 . DOI: 10.11978/j.issn.1009-5470.2010.05.125
Two full-length cDNA sequence encoding pancreatic trypsinogen (TRY) and amylase (AMY) genes were isolated from orange-spotted grouper (Epinephelus coioides) by RT-PCR and RACE methods. The sequences were deposited in the GenBank database under accession number EU715402 and EU715401. The cDNA of TRY was 911bp, encoding 242 amino acids. The deduced amino acid sequence of trypsinogen has a catalytic triad, and sequence analysis reveals that the identities of trypsinogen amino acid are 59.9%–89.7% between fish and mammals. The obtained AMY was 1657bp in length, encoding 512 amino acids. The deduced amino acid of amylase contains eight α-helical and eight β-sheet structures, and the identities of amylase amino acid among fish, mammals and bird are 70.1%–91.8%, which suggests that amylase is relatively more con-served. This research will help to further study the regulation of the two genes and to develop dietary additives to stimulate their expression levels.
Key words： trypsinogen; amylase; gene cloning; sequence analysis; Epinephelus coioides
[1] 尾崎久雄. 鱼类消化生理: 上册[M], 上海: 上海科学技术出版社, 1983.
[2] 荻野珍吉. 鱼类的营养和饲料[M], 北京: 海洋出版社, 1987.
[3] OSSAIN M A R, TAGAWA M, MASUDA R, et al. Changes in growth performance and proximate composition in Japanese flounder during metamorphosis[J]. Fish Biol, 2003, 63: 1283-1294.
[4] WANG S, MAGOULAS C, HICKEY D A. Isolation and characterization of a full-length trypsin-encoding cDNA clone from the Lepidopteran insect, Choristoneura fumi ferana[J]. Gene, 1993, 136 (122): 375-376.
[5] JANACEK S. Sequence similarities and evolutionary relationships of microbial, plant and animal α-amylases[J]. Eur J Biochem, 1994, 224: 519-524.
[6] SUSAN E D, SUZAN M, JEFFREY W G. Molecular analysis of the amylase gene and its expression during development in the winter flounder, Pleuronectes americanus[J]. Aquaculture, 2000, 190: 247-260.
[7] 倪寿文. 草鱼、鲤、鲢、鳙和淀粉酶比较研究[J]. 大连水产学院学报, 1992, 7 (1): 24-31.
[8] SUZUKI T, SRIVASTAVA A S, KUROKAWA T. cDNA cloning and phylogenetic analysis of pancreatic serine proteases from Japanese flounder, Paralichthys olivaceus[J]. Comp Biochem Physiol B, 2002, 131: 63-70.
[9] TITANI K, ERICSSON L H, NEURATH H, et al. Amino acid sequence of dogfish trypsin[J]. Biochem, 1975, (14): 1358-1366.
[10] YOSHINAKA R, SATO M, SUZUKI T, et al. Enzymatic characterization of anionic trypsin of catfish (Parasilurus asotus)[J]. Comp Biochem Physiol B, 1984, 77: 1-6.
[11] MALE R, LORENS J R, SMALKS A O, et al. Molecular cloning and characterization of anionic and cationic variants of trypsin from Atlantic salmon[J]. Eur J Biochem, 1995, 232: 677-685.
[12] SUZUKI T, SRIVASTAVA A S, KUROKAWA T. cDNA cloning and phylogenetic analysis of pancreatic serine proteases from Japanese Bounder, Paralichthys olivaceus[J]. Comp Biochem Physiol B, 2002, 131: 63-70.
[13] COWEY C B, SARGENT J R. NUTRITION. Fish physiology, Vol. VIII[M]//HOAR W S, RANDALL D J, BRETT J R.. New York: Academic Press; 1979: l-69.
[14] NISHIDE T, NAKAMURA Y, EMI M, et al. Primary structure of human salivary α-amylase gene[J]. Gene, 1986, 41: 299-304.
[15] HORII A, EMI M, TOMITA N, et al. Primary structure of human pancreatic a-amylase gene: its comparison with human salivary α-amylase gene[J]. Gene, 1987, 60: 57-64.
[16] GUMUCIO D L, WIEBAUER K, CALDWELL R M, et al. Concerted evolution of human amylase genes[J]. Mol Cell Biol, 1988, 8: 1197-1205.
[17] YOKOUCHI H, HORII A, EMI M, et al. Cloning and characterization of a third type of human α-amylase gene, AMY2B[J]. Gene, 1990, 90: 281-286.
[18] 周景祥, 陈 勇, 黄 权, 等. 鱼类消化酶的活性及环境条件的影响[J]. 北华大学学报: 自然科学版, 2001, 2(1): 70-83.
[19] 余德逛, 王广军, 谢骏, 等. 鞋带石斑鱼消化器官的蛋白酶和淀粉酶活力变化[J]. 浙江海洋学院学报: 自然科学版, 2007, 26(3), 246-251.
[20] BITTERLICH G. Digestive enzyme pattern of two stomachless filter feeders, silver carp, Hypophthamichthys molitrix, and bighead carp, Aristichthys nobiliis[J]. Rich J Fish Biol, 1985, 27: 103-112.
[21] TAKII K, SHIMENO S, TAKEDA M. Changes in digestive enzyme activities in eel after feeding[J]. Bull Jpn Sot Sci Fish, 1985, 51: 2027-2031.
[22] MUNILLA-MORGN R, STARK J R. Metabolism in marine flatfish VI. Effect of nutritional state on digestion in turbot, Scophthulmus maximus (L.)[J]. Comp Biochem Physiol B, 1990, 95: 625-634.
[23] SABAPATHY U, TEO L H. A quantitative study of some digestive enzymes in the rabbitfish, Siganw canaliculatus, and the sea bass, Lates calcarifer[J]. Fish Biol, 1993, 42: 595-602.
[24] LUO Z, LIU Y J,
/
| 〈 |
|
〉 |
