Cloning Of Isoforms Of GLUT1 Gene From Winged Pearl Oyster Pteria Penguin And Their Expression In Response To Glucose Challenge

Facilitated glucose transporter type 1(GLUT1) takes part in transporting glucose in most animal tissues responsible for energy metabolism. To study function of GLUT1 in Pteria penguin, the cDNA sequence of two different isoforms of GLUT1 were obtained from P.penguin transcriptome EST sequence using RACE, the molecular mass of one isoform is 57 kDa and named as PpGLUT1. another isoform is 58 kDa.1. The full length of GLUT1(57kDa) cDNA was 2 068 bp, The gene encoded522 amino acids, 12 transmembrane helical motifs and multiple phosphorylation sites,with N-terminal and C-terminal on the cytoplasmic side, molecular weight of 57.2264 kD, and isoelectric point of 5.17. Fluorescent quantitative PCR results show that PpGLUT1 expressed in all tissues and significantly low in muscle. The high concentration of 0.5g/mL glucose injection result shows that the expression of PpGLUT1 increased within 30 min after injected, and then decreased; at the injection time of 2 h, the expression of PpGLUT1 was lowest; the expression gradually increased after 2 h. Different concentrations of glucose injection test indicate that the expression levels of Pp GLUT1 increased with increase of glucose concentrations ranging from 0.1 to 0.4g/mL and then decreased. The FISH results showed that PpGLUT1 was expressed highly in intestines and rich expression in lamina propria,but low expression in muscular layer.2. The full-length cDNA of GLUT1(58kDa), obtained by RACE strategy, was 2237 bp in length containing an open reading frame(ORF) of 1 584 bp corresponding to a polypeptide of 527 amino acids with estimated molecular mass of 58.9388 kDa and a theoretical isoelectric point of 8.16, a 5′-untranslated region(UTR) of 494 bp and a 3′-UTR of 159 bp with a 29 bp poly(A) tail. SingalP 4.1 analysis revealed that the signal peptide was not detected with 0 as Signal score in this presumed transportprotein. The grand average of hydropathicity(GRAVY) was 0.542, stating that GLUT1(58kDa) was a hydrophobic protein. Blastp protein functional domainanalysis revealed this protein belonged to MFS transporter and showed that the amino acid region from 84 bp to 500 bp is characteristic interval of SP(sugar porter family)model that represent the sugar porter subfamily of the major facilitator superfamily,moreover, the predicted D-xylose transporter XylE and AraJ(arabinose efflux permease) exist in this presumed protein.The deduced amino acid sequence contained five putative N-linked glycosylation sites(NXS/T), including NYTF, NKTI, NKTS,NSTM, NISL, and contained nineteen phosphorylation site with active residues of S(Ser), T(Thr) and W(Tyr) with number of 14, 1and 4, respectively. Topological structure analysis reveal the transporter involve in 12 transmembrane α helical regions and both N-termina and C-termina are in the cytoplasmic, bioinformatics analysis of the deduced polypeptide sequence has identified three significant motifs/domains,which are typically found in the GLUT1 protein of other species: the one consensus sequence is “XXQQXSG”, is present at residue 309-316 aa and located in one transmembrane domain, two other “EXFXQXPR” and “PETKXXTFXEI”, are present at residue 430-437 aa and 490-500 aa, respectively, and they are all situated in cytoplasmic.Based on the above result, GLUT1(57kDa) and GLUT1(58kDa) were expressed mainly in mantle and intestines of Pteria penguin, the two genes will reponse to glucose injection and the regular pattern is similar, we think they have reciprocity in working on physiological mechanism.