1. Molecular Cloning and Differential Gene Expression Analysis of 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) in Andrographis paniculata (Burm. f) Nees.
- Author
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Srinath M, Shailaja A, Bindu BBV, and Giri CC
- Subjects
- Amino Acid Sequence, Andrographis drug effects, Cloning, Molecular, Conserved Sequence, Cyclopentanes pharmacology, Diterpenes metabolism, Escherichia coli metabolism, Genes, Plant, Isopropyl Thiogalactoside pharmacology, Lactones metabolism, Molecular Docking Simulation, Organ Specificity drug effects, Organ Specificity genetics, Oxylipins pharmacology, Plant Proteins genetics, Plant Proteins metabolism, Plant Roots drug effects, Plant Roots metabolism, Protein Domains, Structural Homology, Protein, Transferases chemistry, Transferases metabolism, Andrographis enzymology, Andrographis genetics, Gene Expression Profiling, Gene Expression Regulation, Plant drug effects, Transferases genetics
- Abstract
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.
- Published
- 2021
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