鲈鱼FBP基因的克隆和表达分析

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。

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

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.