Analysis and Comparison of Biochemical and Molecular Structural Characteristics and Phylogenetic Trend of Two h-TypeThioredoxin Isoforms from Grape (Vitis vinifera L. cv. Askari)

Abstract

Thioredoxins (Trxs) are small heat-stable proteins that participate in dithiol-disulfide exchange reactions. In contrast to other organisms, plants contain six different Trx types: f, m, x, y, o and h. The h-type Trx consists of multiple forms that involved in different processes such as cellular protection against oxidative, biotic and abiotic stresses. Two thioredoxin h-type genes, called VvTrxh4 and VvCxxS2, were isolated of grape (Vitis vinifera L. cv. Askari) berry tissue and were cloned into pUC19 plasmid vector. Reverse transcription was carried out using Polymerase Chain Reaction (RT-PCR), to analyze biochemical, structural and phylogenetic characteristics. Nucleotide sequence analysis revealed that the open reading frame of VvTrxh4 and VvCxxS2 genes are 345 bp and 381 bp long, respectively and encode for proteins of 114 and 126 amino acid residues, respectively. Protein sequence analysis showed that VvTrxh4 gene contains a typical catalytic site WCGPC, whereas VvCxxS2 gene harbors the non-typical active site WCIPS. The calculated molecular mass and the predicted isoelectric point of the deduced polypeptides for both VvTrxh4 and VvCxxS2 were 12.76 kDa and 5.22, and also 14.25 kDa and 4.68, respectively. Structural analysis showed that deduced proteins contain a identical 3D structure, whereas phylogenetic study of cloned genes with thioredoxin from other plants revealed that these genes are belonging to different subgroups. In the present research project, VvTrxh4 gene belongs to class IA, while VvCxxS2 gene belongs to class IIIB from h-type thioredoxins

Keywords

• biochemical characteristics
• cloning
• grape
• phylogenetic
• thioredoxin