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| Cloning and Sequence Analysis of Lateolabrax japonicus Glucose-6- phosphatase catalytic subunit (G6PC) cDNA and Its 5'-flanking Region |
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Abstract Glucose-6-phosphatase(G6Pase, EC3.1.3.9) catalyzes the hydrolysis of glucose-6-phosphate into glucose, the final step in the gluconeogenic and glycogenolytic pathways. A full-length cDNA of glucose-6-phosphatase catalytic subunit(G6PC) from Lateolabrax japonicus was amplified by SMART RACE method. The cDNA was 1 651 bp in size, with a 75 bp 5'-UTR, 496 bp 3'-UTR and 1 080 bp ORF, encoding a protein of 359 amino acids with a molecular weight of 40.45 kD and pI 9.30(GenBank accession No. HQ317736.1). The deduced amino acid sequence of L. japonicus G6PC shared high identity with other eleven species, which was from 58% to 85%, and the highest was 85% with Osmerus mordax. L. japonicus G6PC contained three conserved domains. RT-PCR was used to test the expression profile of G6PC in ten tissues, including muscle, eye, spleen, kidney, liver, fat, heart, gill, intestine and brain. The results showed that G6PC was mainly expressed in liver, intestine and kidney, and weakly in brain and gill. A 1 243 bp 5'-flanking region sequence upstream of the translational start of G6PC gene was cloned using genomic walking technique. It contained highly conserved 2 TATA boxes, 2 IRS(insulin response sequence), C/EBPb(CCAAT-enhancer-binding protein b), CRE-BP(cAMP response element binding proteins) and HNF-3b(hepatocyte nuclear factor 3 beta, FoxA2) potential transcriptional factor binding sites. We conclude that L. japonicus G6PC are abundant in liver, intestine and kidney, and the 5'- flanking region of G6PC contains multiple binding sites for transcriptional factor. These results will be helpful foundation for understanding the regulation mechanism of G6PC.
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Received: 21 December 2010
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