摘要
采用RT-PCR和RACE（Rapid Amplification of cDNA Ends）法,分离和克隆翘嘴红鲌（Erythroculter ilishaeformis）磷酸烯醇式丙酮酸羧激酶（PEPCK）基因的全序列,得到2 564bp[不含poly（A）]的全长cDNA,包括111bp5′非翻译区,1 911 bp阅读框以及含Poly（A）信号AATAAA的542 bp3＇非翻译区[不包括Poly（A）].阅读框共编码636个氨基酸,计算的分子量为69.65 kD.该序列含有PEPCK特有的结合草酰乙酯的结构域以及与GTP三磷酸链和Mg^2＋结合的激酶1和2基序.与其他鱼PEPCK的相似性高达83%～96%,和爪蟾、变形虫等其他动物的相似性为50%～69%.[中国水产科学,2006,13（3）：389 396]
Phosphoenolpyruvate carboxykinase （PEPCK; E. C. 4.1.1.32） can catalyse the conversion of oxaloacetate to phosphoenolpyruvate and is an important rate-limiting enzymes involved in hepatic gluconeogenesis. A hypothesis to explain the poor utilisation of dietary glucose by fish is a persistent highly active hepatic glucose production even when the fish were fed with high levels of carbohydrates diets. In mammals, the gluconeogenic enzymes generally correlate with the amount of the corresponding mRNAs and there is decreased gene expression for these hepatic enzymes by feeding carbohydrates. Research revealed gluconeogenic enzymes in rainbow trout is not regulated by carbohydrates. Erythroculter ilishaeformis is a species of carnivorous fish, but in artificial aquaculture situation it can eat carbohydrates feed. Whereas there is no information about gluconeogenic enzymes of Erythroculter ilishaeformis. Here isolation and cloning of the PEPCK were conducted by using RT-PCR and RACE in Erythroculter ilishaeformis. The cDNA was 2 564 bp with 111 bp 5＇UTR, 542 bp 3＇UTR（ excluding poly（A）） and 1 911 bp ORF, which encoded 636 amino acids and had a predicted tool wt of 69.65 kD. The Erythroculter ilishaeforrnis PEPCK appears to possess the PEPCK-specific domain to bind oxaloacetate as well as kinase-1 and kinase-2 motifs to bind the GTP triphosphate chain and Mg^2＋ , respectively. For phylogenetic analysis the deduced amino acid sequence of Erythroculter ilishaeforrnis PEPCK, together with PEPCK sequences reported for other vertebrate and invertebrate species, were aligned by ClustalW, version 1.6. The results revealed the Erythroculter ilishaeformis PEPCK shared 83 % - 96 % sequence identity with those of Tetraodon nigroviridis, rainbow trout and zebrafish, the closest being zebrafish, and shared 50 % - 69 % identity with clawed frog, amoeba, chicken and mouse. But the percent age of identity/similarity was high in the region of PEPCK specific domain, kinase 1 and kinase2 motifs. This confirms that this se
出处
《中国水产科学》
CAS
CSCD
北大核心
2006年第3期389-396,共8页
Journal of Fishery Sciences of China
基金
国家重点基础研究"973"规划项目(2004CB117401)
