Isolation of 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of methyl erythritol diphosphate (MEP) pathway, in silico analysis and differential tissue specific ApHDR expression in Andrographis paniculata (Burm. f) Nees

Abstract

The full length Andrographis paniculate 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of MEP pathway was isolated for the first time. The ApHDR ORF with 1404 bp flanked by 100 bp 5′UTR and 235 bp 3′UTR encoding 467 amino acids (NCBI accession number: MK503970) and cloned in pET 102, transformed and expressed in E. coli BL21. The ApHDR protein physico-chemical properties, secondary and tertiary structure were analyzed. The Ramachandran plot showed 93.8% amino acids in the allowed regions, suggesting high reliability. The cluster of 16 ligands for binding site in ApHDR involved six amino acid residues having 5–8 ligands. The Fe-S cluster binding site was formed with three conserved residues of cysteine at positions C123, C214, C251 of ApHDR. The substrate HMBPP and inhibitors clomazone, paraquat, benzyl viologen’s interactions with ApHDR were also assessed using docking. The affinity of Fe-S cluster binding to the cleft was found similar to HMBPP. The HPLC analysis of different type of tissue (plant parts) revealed highest andrographolide content in young leaves followed by mature leaves, stems and roots. The differential expression profile of ApHDR suggested a significant variation in the expression pattern among different tissues such as mature leaves, young leaves, stem and roots. A 16-fold higher expression of ApHDR was observed in the mature leaves of A. paniculata as compared to roots. The young leaves and stem showed 5.5 fold and fourfold higher expression than roots of A. paniculata. Our result generated new genomic information on ApHDR which may open up prospects of manipulation for enhanced diterpene lactone andrographolide production in A. paniculata.

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Acknowledgements

Authors would like to thank DST-PURSE-II sponsored by Department of Science and Technology (DST) New Delhi, and OU-UGCCPEPA, UGC-BSR-RFSMS sponsored by University Grants Commission (UGC) New Delhi for financial support and fellowship to AS, MS, BBVB.

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Shailaja, A., Srinath, M., Bindu, B.V.B. et al. Isolation of 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of methyl erythritol diphosphate (MEP) pathway, in silico analysis and differential tissue specific ApHDR expression in Andrographis paniculata (Burm. f) Nees. Physiol Mol Biol Plants 27, 223–235 (2021). https://doi.org/10.1007/s12298-021-00952-0

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Keywords

  • Andrographis paniculata
  • HDR
  • Gene isolation
  • Andrographolide
  • qRT-PCR
  • Tissue specific gene expression