Molecular Cloning and Differential Gene Expression Analysis of 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) in Androgra
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ORIGINAL PAPER
Molecular Cloning and Differential Gene Expression Analysis of 1‑Deoxy‑D‑xylulose 5‑Phosphate Synthase (DXS) in Andrographis paniculata (Burm. f) Nees Mote Srinath1 · Aayeti Shailaja1 · Byreddi Bhavani Venkata Bindu1 · Charu Chandra Giri1 Accepted: 17 November 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords Andrographis paniculata · 1-deoxy-D-xylulose 5-phosphate synthase · Elicitation · Andrographolide · Adventitious root cultures · QRT-PCR · In silico docking
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12033-020-00287-3) contains supplementary material, which is available to authorized users. * Charu Chandra Giri [email protected] Mote Srinath [email protected] Aayeti Shailaja [email protected] Byreddi Bhavani Venkata Bindu [email protected] 1
Centre for Plant Molecular Biology, Osmania University, Hyderabad 500007, Telangana, India
Isoprenoids are the important diverse group of bioactive compounds performing vital biological functions in plants, bacteria, and mammals. Especially in plants, they perform significant roles such as redox reactions, light harvesting, growth regulation, membrane structure, and development [1]. All isoprenoids are synthesized by two 5-carbon monomers, isopentenyl pyrophosphate (IPP), and its isomer, di
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