Molecular characterisation of 3-hydroxy-3-methyl glutaryl CoA reductase (hmgr) gene from solanaceous plants

Abstract

3-Hydroxy-3-methyl coenzyme A reductase (HMGR) is a key enzyme in the synthesis of mevalonate, which is the precursor of terpenoid compounds, that are vital in biological processes like respiration, photosynthesis, growth, reproduction and defense mechanisms in plants. It is also the rate-limiting enzyme in tri- and sesqui-terpene biosynthesis. The gene encoding HMGR protein has been cloned and sequenced in several plants like Arabidopsis, rubber, potato, tobacco, tomato, wheat, rice, apple, periwinkle, chilli and black pepper. In the present study, an attempt was made to isolate and characterize hmgr gene from four plants belonging to family Solanaceae: Solanum xanthocarpum, S. nigrum, S. torvum and Physalis minima, having medicinal/ insecticidal properties. Gene was amplified through polymerase chain reaction with two pairs of gene-specific primers, designed on the basis of conserved boxes in HMGR from other solanaceous plant species. The amplified products were cloned in the plasmid vector pGEMT and sequenced. A total of four sequences were characterized: two from S. torvum (945 and 1835bp) and one each from S. xanthocarpum (882bp) and S. nigrum (945bp). Theoretical analysis using various tools revealed conserved domains corresponding to HMGR in all the four sequences. The sequences were found to be rich in glycine, which is important in maintaining the correct structure of -domain. These sequences exhibited a high degree of identity with hmgr genes in tomato, tobacco and potato, all belonging to family Solanaceae. Generally, HMGRs exhibit a high level of homology at the catalytic domain, present at the C-terminal region. Two functional domains (CoA reductase and NAD binding) could be located on Sxhmgr, the sequence from S. xanthocarpum. However, no putative transmembrane region could be located in any of the sequences, probably because the N-terminal region has not been represented in the cloned sequence. Normally, two transmembrane regions are associated with plant HMGRs, involved in anchoring the enzyme to endoplasmic reticulum. Attempts to obtain 5’ and 3’ ends of Sxhmgr did not prove successful. Sequence diversity of hmgr gene among various plat species was determined. The evolutionary distance of Sxhmgr was much lower, compared to other sequences. The two sequences cloned from S. torvum showed divergence, indicating that hmgr genes could be divergent, within the species. For further exploitation of the sequences in genetic improvement programmes, the full-length genes will have to be cloned from the mRNA population and expressed in Escherichia coli. The expression profile of mRNA in different parts and at different developmental stages of the plant will also throw light on the role of these genes in various metabolic processes taking place.