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cDNA cloning; 3D structure; Gracilaria fisheri; L-lectin; RACE-PCR

A legume-type lectin (L-lectin) gene of the red algae Gracilaria fisheri (GFL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of GFL was 1714 bp and contained a 1542 bp open reading frame encoding 513 amino acids with a predicted molecular mass of 56.5 kDa. Analysis of the putative amino acid sequence with NCBI-BLAST revealed a high homology (30–68%) with legume-type lectins (L-lectin) from Griffithsia japonica, Clavispora lusitaniae, Acyrthosiphon pisum, Tetraodon nigroviridis and Xenopus tropicalis. Phylogenetic relationship analysis showed the highest sequence identity to a glycoprotein of the red algae Griffithsia japonica (68%) (GenBank number AAM93989). Conserved Domain Database analysis detected an N-terminal carbohydrate recognition domain (CRD), the characteristic of L-lectins, which contained two sugar binding sites and a metal binding site. The secondary structure prediction of GFL showed a 𝛽-sheet structure, connected with turn and coil. The most abundant structural element of GFL was the random coil, while the 𝛼-helixes were distributed at the N- and C-termini, and 21 𝛽-sheets were distributed in the CRD. Computer analysis of three-dimensional structure showed a common feature of L-lectins of GFL, which included an overall globular shape that was composed of a 𝛽-sandwich of two anti-parallel 𝛽-sheets, monosaccharide binding sites, were on the top of the structure and in proximity with a metal binding site. Northern blot analysis using a DIG-labelled probe derived from a partial GFL sequence revealed a hybridization signal of ∼1.7 kb consistent with the length of the full-length GFL cDNA identified by RACE. No detectable band was observed from control total RNA extracted from filamentous green algae.

Sukanya Suttisrisung¹ Saengchan Senapin^{1 2} Boonsirm Withyachumnarnkul^{1 3 4} Kanokpan Wongprasert⁴

1. Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand
2. National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand
3. Shrimp Genetic Improvement Center, National Center for Genetic Engineering and Biotechnology, Science and Technology Development Agency, Surat Thani, Thailand
4. Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand

Published

December 2011

Manuscript received

21 March 2011

Accepted

22 July 2011

Early published

30 September 2011