鲈鱼FBP基因的克隆和表达分析

海洋学研究 ›› 2012, Vol. 30 ›› Issue (4): 78-83.

鲈鱼FBP基因的克隆和表达分析

童彩环, 钱云霞^*, 郑伟贤, 韩柳

宁波大学生命科学与生物工程学院,应用海洋生物技术教育部重点实验室,浙江宁波 315211

收稿日期:2011-11-25 修回日期:2011-12-28 出版日期:2012-12-15 发布日期:2022-11-30
通讯作者: *钱云霞,教授,E mail:qianyunxia@nbu.edu.cn
作者简介:童彩环(1986-),女,江西高安市人,硕士研究生,主要从事水产分子生物学方面的研究。E-mail: tongcaihuan@163.com
基金资助:
国家自然科学基金资助项目(30671608); 浙江省科技厅创新团队资助项目(2010R50029)

Molecular cloning and tissue-specific expression of FBP of Lateolabrax ja ponicus

TONG Cai-huan, QIAN Yun-xia^*, ZHENG Wei-xian, HAN Liu

Key Laboratory of Applied Marine Biotechnology of the Ministry of Education, Facultyof Life Science and Biotechnology,Ningbo University, Ningbo 315211, China

Received:2011-11-25 Revised:2011-12-28 Online:2012-12-15 Published:2022-11-30

摘要/Abstract

摘要： 果糖-1,6-二磷酸酶(fructose-1,6-bisphosphatase,FBP, EC 3. 1.3.11)可催化果糖-1,6-二磷酸水解成果糖-6-磷酸和无机磷酸盐,是糖异生途径中的关键酶之一。本研究运用SMART RACE技术从鲈鱼Lateolabrax japonicus肝脏中分离克隆了FBP基因的全长cDNA序列,该基因全长1 357 bp,其中5’非翻译区和3’非翻译区分别为42 bp和301 bp,开放阅读框为1 014 bp,共编码337个氨基酸。蛋白质分子量约为36.7 kD,理论pI为6.90。氨基酸序列分析表明,鲈鱼FBP与其它动物的肝脏型FBP相似性很高,与裸盖鱼、彩虹胡瓜鱼、斑马鱼、异育银鲫和大西洋鲑的肝脏型FBP的同源性分别为94.3%,90. 8%,89.3%,88.1%和84.1%。系统发育分析显示,鲈鱼FBP与其它鱼类的肝脏型FBP成簇后再与哺乳动物的肝脏型FBP聚成一支,然后才与哺乳动物的肌肉型FBP汇成簇。同时用RT-PCR分析了FBP基因在鲈鱼肝脏、肌肉、心脏、眼、肠、肾脏、脂肪、脾脏、鳃和大脑等10个组织的表达,结果表明FBP仅在肝脏、肾脏和肠这3个组织中有较高的表达,与糖异生发生组织基本一致,因此推测该FBP属于肝脏型。

关键词: 果糖-1,6二磷酸酶, 鲈鱼, 克隆, 表达分析

Abstract: Fructosel, 6-bisphosphatase ( EC 3. 1. 3. 11) can catalyse the hydrolysis of fructose-l, 6-bisphosphate into fructose-6-phosphate and inorganic phosphate. It is one of the key enzymes in the gluconeogenic pathway. The full-length 1 357 bp FBP cDNA was isolateded from the liver of sea perch Lateolabrax japonicus with SMART RACE technology. It consists of a 42 bp5' UTR,a 301 bp 3' UTR and an open reading frame (ORF) of 1 014 bp,encoding a polypeptide of 337 amino acids with a predicted molecular weight of 36. 7 kD and electric point of 6. 90. Sequencing analysis of amino acid shows that the Lateolabrax japonicus FBP has high amino acid homology with the liver-type FBP gene from other species, and the homology is 94. 3% with Anopiopoma fimbria, 90. 8% with Osmerus mordax,89. 3% with Danio rerio, 88. 1% with Carassius gibelio and 84. 1% with Salmo salar. Phylogenetic analysis shows that Lateolabrax japonicus FBP clustered with liver-type FBP of fish, then with liver-type FBP of mammals, and still then clustered with muscle-type FBP of mammals to form a family. Semi-quantitative RT-PCR was used to determine the expression profiles of FBP from liver, muscle, heart, eye,intestine, kidney, fat, spleen, gill and brain. The results show that FBP was expressed only in liver, intestine and kidney. This conclusion is in consistent with the fact that liver, kidney and intestine are the major gluconeogenic tissues of teleost. These results indicate that the FBP obtained is of liver-type.

Key words: Fructose-1,6-bisphosphatase, Lateolabrax japonicus, cloning, expression

中图分类号:

童彩环, 钱云霞, 郑伟贤, 韩柳. 鲈鱼FBP基因的克隆和表达分析[J]. 海洋学研究, 2012, 30(4): 78-83.

TONG Cai-huan, QIAN Yun-xia, ZHENG Wei-xian, HAN Liu. Molecular cloning and tissue-specific expression of FBP of Lateolabrax ja ponicus[J]. Journal of Marine Sciences, 2012, 30(4): 78-83.

参考文献

[1] PILKISS J, CLAUS T H. Hepatic gluconeogenesis/glycolysis: regulation and structure/function rela tionships of substrate cycle enzymes[J]. Annu Rev Nutr, 1991,11:465-515.
[2] RAKUS D, MACIASZCZYK E, WAWRZYCKA D, et al. The origin of the high sensitivity of muscle fructosel, 6-bis phos phatase towards AMP[J]. FEBS Letters, 2005,579(25):5 577-5 581.
[3] LU G, STEC B, GIROUX E L, et al. Evidence for an active Tstate pig kidney fructose 1, 6-bisphosphatase: Interface residue Lys-42 is important for allosteric inhibition and AMP cooperativi-ty[J]. Protein Science, 1996,5(11):2 333-2 342.
[4] TILLMANN H, BERNHARD D, ESCHRICH K.Fructose 1,6-bisphosphatase genes in animals[J]. Gene,2002, 291(1/2):57-66.
[5] SOULIMANE T.Thermus thermophilus encodes an archaeal-like fructose-1, 6-bisphosphatase: Purification of native and recombi-nant protein for structural studies[J]. Protein Expression and Pur-ification, 2010,74(2):175-180.
[6] SKALECKI K, RAKUS D, WISNIEWSKI J R,et al. cDNA se-quence and kinetic properties of human lung fructose (1, 6) bi-sphosphatase[J]. Arch Biochem Biophys, 1999,365(1):1-9.
[7] AL ROBAIY s, ESCHRICH K. Rat muscle fructose-1, 6-bi-sphosphatase: Cloning of the cDNA, expression of the recombi-nant enzyme, ande xpression analysis in different tissues[J]. Biol Chem,1999,380(9):1 079-1 085.
[8] HEBEISEN P, HAAP W, KUHN B, et al. Orally active amin-opyridines as inhibitors of tetrameric fructose-1, 6-bi sphosphatase[J]. Bioorganic &. Medicinal Chemistry Letters, 2011,21(11): 3 237-3 242.
[9] KIRCHNER S, PANSERAT S, LIM P L, et al. The role of he-patic,renal and intestinal gluconeogenic enzymes in glucose home-ostasis of juvenile rainbow trout[J]. J Comp Physiol B, 2008,178(3):429-438.
[10] QIAN Yun-xia, YANG Sun-xiao, TONG Lrjuan, et al. Low sa-linity induces cytosolic PEPCK expression in Sea Perch (Lateo-labrax ja ponicus)[J]. Chinese Journal of Biochemistry and Mo-lecular Biology, 2006,26(7):651-658.
钱云霞,杨孙孝,童丽娟,等.低盐度可诱导鲈鱼胞浆型PEPCK基因表达[J].中国生物化学与分子生物学报,2006,26(7):651-658.
[11] QIAN Yun-xia, ZHENG Wei-xian, SONG Juan-juan. Cloning and sequence analysis of Lateolabrax ja ponicus Glucose 6-phos-phatase Catalytic Subunit (G6PC) cDNA and its 5'-flanking re-gion[J]. Journal of Agricultural Biotechnology, 2011, 19(4):606-615.
钱云霞,郑伟贤,宋娟娟.鲈鱼6-磷酸葡萄糖酶催化亚基(G6PC)cDNA和5’侧翼序列的克隆及分析[J].农业生物技术学报, 2011,19(4):606-615.
[12] GIZAK A,MAJKOWSKI M, DUS D, et al. Calcium inhibits muscle FBPase and affects its intracellular localization in cardio-myocytes[J]. FEBS Lett, 2004, 576(3):445-448.
[13] RAKUS D, TILLMANN H, WYSOCKI R, et al. Different sen-sitivities of mutants and chimeric forms of human muscle and liv-er fructose 1, 6-bisphosphatases towards AMP[J]. Biol Chem, 2003,384(1):51-58.
[14] MITHIEUX G, GAUTIER STEIN A, RAJASF, et al. Contri-bution of intestine and kidney to glucose fluxes in different nutri-tional states in rat[J]. Comp Biochem Physiol B Biochem Mol Biol,2006,143(2):195-200.
[15] SUAREZ R K, MOMMSEN T P.Gluconeogenesis in teleost fi-shes[J]. CanJ Zool, 1987,65:1 869-1 882.
[16] WANG Rui, XIAO Qing, GUI Jian-fang. Molecular cloning and expression analysis of fructose 1, 6 bisphosphatase in gibel carp[J]. Acta Hydrobiologica Sinica, 2010,34(6):1 130-1 135.
王锐,肖青,桂建芳.银鲫果糖-1,6-二磷酸酶的分子克隆与表达分析[J].水生生物报,2010,34(6):1 130-1 135.
[17] TILLMANN H, STEIN s, LIEHR T, et al. Structure and chromosomal localization of the human and mouse muscle fruc-tose-1, 6-bisphosphatase genes[J]. Gene, 2000, 247(1/2):241-253.