In silico characterization and differential expression pattern analysis of conserved HMG CoA reductase domain isolated from Aconitum balfourii Stapf

Abstract

The 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) is the key enzyme of mevalonate pathway in plants. A partial genomic DNA fragment encoding HMGR conserved domain (denoted as AbHMGR) is isolated from Aconitum balfourii Stapf. It comprises 871 bp encoding 290 amino acids. In silico analysis reveals that it had extensive similarities to other plant HMGR gene. Domain analysis of AbHMGR showed two highly conserved NADPH and HMG CoA domains. Docking study predicted inhibitor, substrate and cofactor binding sites in the protein. Expression analysis revealed that AbHMGR is similarly expressed in all tested tissues with differential pattern. The highest expression was found in leaf tissue. However, fold expression in root and shoot tissue was almost similar. Enzyme activity of HMGR was found to be much higher in leaf tissue as compared to other tissues. The highest aconitine content (0.015 %) was obtained in root tissues. Our data laid a foundation for further investigation of HMGR role in Aconitum balfourii.

Keywords: 3-Hydroxy-3-methyl glutaryl-CoA coenzyme A reductase; Aconitine; Expression profiling; HMGR; Mevalonate pathway.

Fig. 1

Phylogenetic tree of AbHMGR proteins from different plant species. Sequence analysis was performed using ClustalW and the UPGMA method was applied to create trees. AbHMGR (AGK24692.1) and related proteins from Panax quinquefolius (ACV65036.1), Ricinus communis (XP_002510732.1), Solanum lycopersicum (AL16927.1), Morus alba (AAD03789.1), Litchi chinensis (ABF56518.2), Dimocarpus longan (AET72044.1), Eucommia ulmoides (AV54051.1), Coffea arabica (ADR51242.1), Gentiana macrophylla (AFN89599.1), Linum usitatissimum (ACN38874.1), Hevea brasiliensis (BAF98280.1), Bacopa monnieri (ADX01170.1), Artemisia annua (AAA68966.1), Catharanthus roseus (AAT52222.1), Salvia miltiorrhiza (ACD37361.1), Camellia sinensis (AHB64333.1), Gentiana lutea (BAE92730.1), Vitis vinifera (CBI40773.3), Gossypium arboreum (KHG04251.1), Arabidopsis thaliana (NP_177775.2), Oryza sativa Japonica group (Os08g0512700)

Fig. 2

Distribution of motifs among 22 sequences subjected to MEME

Fig. 3

Docking study of AbHMGR protein of A. balfourii Stapf. a The modeled overall 3D structure of AbHMGR, b AbHMGR docked with NADP(H), b AbHMGR docked with mevinolin, c AbHMGR docked with CoA. The bindings of all three ligands are indicated

Fig. 4

a Expression pattern of AbHMGR in different tissues of A. balfourii Stapf. Total RNA was isolated from leaves, roots and stems, respectively, was subjected to real-time PCR amplification (upper panel). Actin gene was used as the control to show the normalization of the templates in PCR reactions (lower panel). b Relative expression of AbHMGR in different tissues using real-time PCR. Final data are represented in graphical form. Three independent determinations for each parameter were recorded and mean ± SE values were calculated. CRD were used for analyzing gel data and real-time data

Fig. 5

Graphical form of Z score transformation analysis for correlation study among AbHMGR gene expression, enzyme activity and percent aconitine in different tissues of A. balfourii Stapf