Molecular Cloning and Differential Gene Expression Analysis of 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) in Andrographis paniculata (Burm. f) Nees.

Andrographis paniculata 1-deoxy-D-xylulose-5-phosphate synthase (ApDXS) gene (GenBank Accession No MG271749.1) was isolated and cloned from leaves for the first time. Expression of ApDXS gene was carried out in Escherichia coli Rosetta cells. Tissue-specific ApDXS gene expression by quantitative RT-PCR (qRT-PCR) revealed maximum fold expression in the leaves followed by stem and roots. Further, the differential gene expression profile of Jasmonic acid (JA)-elicited in vitro adventitious root cultures showed enhanced ApDXS expression compared to untreated control cultures. A. paniculata 3-hydroxy-3-methylglutaryl-coenzyme A reductase (ApHMGR) gene expression was also studied where it was up-regulated by JA elicitation but showed lower expression compared to ApDXS. The highest expression of both genes was found at 25 µm JA elicitation followed by 50 µm. HPLC data indicated that the transcription levels were correlated with increased andrographolide accumulation. The peak level of andrographolide accumulation was recorded at 25 µM JA (9.38-fold) followed by 50 µM JA (7.58-fold) in elicitation treatments. The in silico generated ApDXS 3D model revealed 98% expected amino acid residues in the favored and 2% in the allowed regions of the Ramachandran plot with 92% structural reliability. Further, prediction of conserved domains and essential amino acids [Arg (249, 252, 255), Asn (307) and Ser (247)] involved in ligand/inhibitor binding was carried out by in silico docking studies. Our present findings will generate genomic information and provide a blueprint for future studies of ApDXS and its role in diterpenoid biosynthesis in A. paniculata.

Isolation, characterization and in silico analysis of 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) gene from Andrographis paniculata (Burm. f) Nees.

3-Hydroxy-3-methylglutaryl-coenzymeA reductase (HMGR), the first rate-limiting enzyme of Mevalonate (MVA) pathway was isolated from Andrographis paniculata (ApHMGR) and expressed in bacterial cells. Full length ApHMGR (1937 bp) was submitted to NCBI with accession number MG271748.1. The open reading frame (ORF) was flanked by a 31-bp 5'-UTR, 118-bp 3'-UTR and ApHMGR contained a 1787 bp ORF encoding protein of 595 amino acids. ApHMGR protein was approximately 64 kDa, with isoelectric point of 5.75. Isolated ApHMGR was cloned into pET102 vector and expressed in E. coli BL21 (DE 3) cells, and characterized by SDS-PAGE. HPLC analysis for andrographolide content in leaf, stem and root of A. paniculata revealed highest in leaf tissue. The expression patterns of ApHMGR in different plant tissues using qRT-PCR revealed high in root tissue correlating with HPLC data. Three dimensional (3D) structural model of ApHMGR displayed 90% of the amino acids in most favored regions of the Ramachandran plot with 93% overall quality factor. ApHMGR was highly conserved with plant specific N-terminal membrane domains and C-terminal catalytic regions. Phylogenetic analysis showed A. paniculata sharing common ancestor with Handroanthus impetiginosus. 3D model of ApHMGR was screened for the interaction with substrates NADPH, HMG CoA and inhibitor using Auto Dock Vina. In silico analysis revealed that full length ApHMGR had extensive similarities to other plant HMGRs. The present communication reports the isolation of full length HMGR from A. paniculata, its heterologous expression in bacterial cells and in silico structural and functional characterization providing valuable genomic information for future molecular interventions.