Cloning and characterization of Myo-inositol phosphate synthase gene (dlMIPS) and analysis of the putative structure of the enzyme responsible for the accumulation of anti-nutrient phytate in dolichos bean (Dolichos lablab L.)

Abstract

The major storage form of phosphorus in mature cereal and legume seeds is phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, InSP6) which binds metallic cations such as calcium, zinc, magnesium and iron to form a mixed salt called phytate. Thus, phytates act as anti-nutrients, rendering the minerals unavailable. Myo-inositol phosphate synthase (MIPS) is the major gene for phytate synthesis in legumes and it is characterized only in few major legumes such as soybean, green gram, black gram, cowpea and french bean. Even though dolichos bean is the legume with the highest content of phytic acid (1000–1350 mg/100 g), MIPS gene from this important crop is not characterized yet. For the strategies such as RNA mediated gene silencing, which is potential enough to reduce the level of phytates in crops, mRNA sequence should be completely known. For this purpose, RNA was isolated from the developing seeds and dolichos MIPS cDNA was isolated using one-step RT-PCR system with high fidelity DNA polymerase. From this, dlMIPS gene was cloned and sequence characterized. Gene dlMIPS comprised of 1776 bp with an ORF of 1620 bp, which encodes 539 amino acids. Phylogenetic analysis of the sequence had shown that dolichos bean is closer to green gram, cowpea and french bean. Secondary and tertiary structures of the enzymes from dolichos bean, cowpea and french bean were predicted through homology modeling and compared. Subsequent prediction and comparative analysis of the active sites of enzyme from these legumes had shown that the ligand binding residues vary among legumes.