Characterization of the glucose-6-phosphate isomerase (GPI) gene from the halotolerant alga Dunaliella salina.
Although glucose-6-phosphate isomerase (GPI) plays an important role in glycolysis of both the prokaryotes and eukaryotes, studies on the GPI have not been involved in the halotolerant, unicellular green alga Dunaliella salina (D. salina). In this study, a 2,338 bp of full-length cDNA cloned using rapid amplification of cDNA end (RACE) technique contained an open reading frame (ORF) of 1,980 bp encoding 660 amino acids, which has a predicted molecular weight of 73.3 kD and pI of 6.22 and shares high homology with other organisms. The cloned full-length cDNA was heterologously expressed in Escherichia coli and the recombinant GPI proteins purified using Ni-NTA His Bind column were consistent with the anticipated size of ~75 kD. Predicted 2D and 3D structures of GPI proteins possessed potential active motifs including "GEPGTNGQHSFYQLIHQG" and "VQGFIWGINSFDQWGVELGK", and critical active site residues, such as Ser 241, Ser 296, Thr 298, Thr 301, Arg 358, Glu 444, His 475 and Lys 600. Real time quantitative RT-PCR demonstrated that the expression level of the GPI gene from D. salina (DsGPI) was induced by 3.5 M NaCl with 14-fold higher than that by 1.5 M NaCl (P < 0.01), but inhibited by the light with 4-fold lower than that in the dark (P < 0.05). It is concluded that the cloned GPI gene is indeed from D. salina and may respond to salt and light.
Cui
Liuqing
L
Institute of Tumor Molecular Surgery, The First Affiliated Hospital, Zhengzhou University, 40 Daxue Road, 450052, Zhengzhou, China.
Xue
Lexun
L
Li
Jie
J
Zhang
Lei
L
Yan
Hongxia
H
D000064:Acclimatization / Q000187:drug effects / Q000235:genetics; D020418:Algal Proteins / Q000737:chemistry / Q000235:genetics; D000460:Chlorophyta / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics*; D006219:Halogens / Q000494:pharmacology; D008027:Light; D008958:Models, Molecular; D010802:Phylogeny; D011994:Recombinant Proteins / Q000302:isolation & purification; D055051:Salt-Tolerant Plants / Q000201:enzymology / Q000235:genetics
D020418:Algal Proteins; D018076:DNA, Complementary; D006219:Halogens; D011994:Recombinant Proteins; D005956:Glucose-6-Phosphate Isomerase
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