Cloning and comparative studies of seaweed trehalose-6-phosphate synthase genes

Abstract

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.

Keywords: RACE-PCR; comparative genomics of TPS genes; gene cloning; seaweed; trehalose-6-phosphate synthase gene.

Figure 1

Nucleotide and deduced amino acid sequences of the PyTPS gene. The start and stop codons are shown in boldface. The amino acid residues 603–611 and 795–804 are the two typical conserved regions in the phosphatase family and are shaded here with a gray background.

Figure 2

Over-expression of the PyTPS gene in E. coli strain BL21(DE3). 15 μL of cell lysate was fractionated in a SDS/7.5% polyacrylamide gel. Lane 1, cell lysate of E. coli transformed by pET22b; Lane 2, cell lysate of E. coli transformed by pET22b/PyTPS; M, molecular mass markers (Sigma). The gel was stained with Coomassie Brilliant Blue. Arrow points to the overexpressed PyTPS protein at ~101 kDa.

Figure 3

Block diagram of the TPS proteins from P. yezoensis, rice (O. sativa) and bacteria (E. coli). The N-terminal TPS domain is marked as the light-colored boxes (left side), and the C-terminal TPP domain is marked as the dark-colored boxes (right). From top to bottom: the corresponding domains of the TPS proteins from P. yezoensis, E. coli and O. sativa.

Figure 4

Dendrogram analysis of TPS proteins from five different organisms. The PyTPS (AAW27916) is compared with OsTPS (deduced from AAT01318.1), AtTPS5 (deduced from BAC43297.1), ScTPS2 (deduced from CAA50025.1) and EcOtsAB (deduced from NP_288332.1 and NP_288333.1).

Figure 5

Dendrogram analysis of 10 seaweed TPS genes using the DNAMAN program. See Table 2 for nomenclature of the TPS genes in detail.

Figure 6

Multiple alignment of the amino acid sequence of the TPS proteins from 10 seaweed species. The dots indicate positions where identical amino acids were observed between the 10 seaweed TPS proteins. Varied amino acid residues are shown with their location numbers.

Figure 7

Polygenetic analysis of the 10 seaweed TPS proteins.