Cloning and characterisation of myo-inositol phosphate synthase gene coding for phytates in dolichos lablab L.

Abstract

Hyacinth bean or dolichos bean (Dolichos lablab L.) is a major vegetable legume grown in the tropical and subtropical regions of the world. Legumes are considered as the major source of protein for countries having short supplies of animal protein. The major storage form of phosphorus (P) in mature cereal and legume seed is phytic acid (myo- inositol-1,2,3,4,5,6-hexakisphosphate, InSP6) which strongly binds metallic cations such as calcium, zinc, magnesium and iron to form a mixed salt called phytate. Thus, the phytates are characteristic anti-nutrients, rendering the minerals unavailable. Dolichos bean has a high content of phytic acid (1000-1350 mg/ 100 g). The Myo-inositol phosphate synthase (MIPS) is the major gene for phytate synthesis in legumes. Suppression of MIPS will help in generating lines with reduced phytate content, for which, sequence characterization of MIPS is mandatory. Thus, this research work was carried out with the objective to sequence and annotate myo-inositol phosphate synthase gene from the cDNA of developing dolichos bean seeds.

Good quality total RNA from the developing seeds (3-5 days after seed set) of Dolichos lablab (var. Hima) was isolated using Purelink® Plant RNA Purification Reagent (Invitrogen). The total RNA was subjected to cDNA synthesis and PCR amplification using SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase (Invitrogen) in which reverse transcription and PCR occured in a single tube. The PCR primers were designed from the coding regions of MIPS sequence of Vigna radiata, retrieved from NCBI. As cloning of a full-length gene is difficult, primers were designed as two sets of overlapping sequences of a full-length gene, namely MIPS V F - M1R (880 bp) and M2F – MIPS V R (900 bp) combinations. The purified PCR products were ligated into pTZ57R/T and cloned in E. coli DH5α. The recombinant clones were analyzed by blue or white screening. The transformation was confirmed by colony PCR, plasmid was isolated and used for sequencing.

The DNA sequence was subjected to homology search using BLASTn and it had showed 96 per cent similarity and 98 per cent query coverage with the MIPS sequence of Vigna unguiculata. The 1776 bp cDNA with 1620 bp coding sequence encoded for a protein of 539 amino acids as predicted by ORF FINDER software. The prediction of a 3D- protein structure of the deduced MIPS sequences of D. lablab, V.unguiculata and P.vulgaris were performed by MODELLER 9.14 and was visualized using RASMOL. Similarly, the prediction and comparative analysis of the active sites of enzyme from D. lablab, V.unguiculata and P.vulgaris had shown that the ligand binding residues varies among different species of the same protein.The deduced protein sequences of MIPS used as a query in BLASTp search showed similarity of 49.5 per cent with A chain of MIPS sequence of Caenorhabditis elegans (1VKO A). The constructed structure was further analyzed in Ramachandran plot using the RAMPAGE server and the results showed that 490 residues were present in the favoured region and only 4 residues were present in the outlier region. The phylogenetic relationship of the lablab MIPS sequences with that of other legumes was analyzed by neighbor-joining method using MEGA.v.10.0.5. It was closer to the MIPS gene of Vigna radiata, Vigna unguiculata and Phaseolus vulgaris, which was in line with the BLASTn results.

This is the first study in dolichos bean to characterize its MIPS gene. The information generated in this study could be directly used in RNAi and similar cisgenic or gene editing strategies to generate lablab accessions with reduced phytic acid content.