Cloning and functional assessment of the protein disulfide isomerase (PDI-V) gene and its promoter sequences from Haynaldia villosa


Abstract: Protein disulfide isomerase (PDI) and PDI-like genes encode key foldase enzymes that play an important role in disulfide bond formation, isomerization, and many other metabolic functions in plants. Our previous results indicate that the PDI-V expression is tissue-specific and that it is upregulated by powdery mildew and other abiotic stress treatments. In the current study, the complete PDI-V gene and its promoter sequence were cloned to identify gene structure and the important cis-regulating motifs responsible for various genes' activities. Sequence analysis revealed the size of the cloned genomic sequence and promoter region as 5728 bp and 2086 bp, respectively. The PDI-V gene had nine exons and eight introns; this showed a highly conserved gene structure with that of wheat TaPDIL-5 in terms of exon size and number of exons/introns. The phylogenetic analysis of PDI-V with orthologue sequences from the A, B, and D genome of wheat, barley, and rice clearly demonstrated that the gene was highly conserved across the grass family. In silico analysis of the PDI-V promoter revealed a large number of elements responsible for endosperm or embryo specific expression, and for defense or stress responses against pathogens including fungi and other abiotic stresses such as high or low temperature and drought. Transient expression of the GUS gene in wheat leaf epidermal cells under the control of PDI-V promoter proved its functionality. However, no significant variation was observed in the number of epidermal cells having GUS activity between powdery mildew inoculated and noninoculated wheat leaves, which suggests that the 5'-untranslated region may also play a critical role in promoter activity. This point requires further exploration.

Keywords: Haynaldia villosa, protein disulfide isomerase, cloning, promoter, in silico analysis

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