Abstract

Proteomic analysis offers a new approach to identify a broad spectrum of genes that are expressed in living system. We applied this technique to investigate the protein changes under in vitro and in vivo conditions, since in vitro cultures is considered to be an alternative approach to traditional agriculture in the industrial production of the biomolecules. To better understand the proteins and enzymes involved in withanolide biosynthetic pathway, detailed two-dimensional gel electrophoresis (2-DE) and mass spectrometric analysis of in vitro grown adventitious roots and in vivo root samples of Withania somnifera were conducted. Of ~55 protein spots resolved in the two-dimensional gels, 35 protein spots were similarly expressed in both in vitro and in vivo root tissues and nine protein spots were differentially expressed only in in vitroroot tissue and could be reproducibly displayed across an isoelectric focusing range of 4 to 7. A total of 44 protein spots (both in vitro and in vivo) were analyzed by matrix-assisted laser desorption ionization time of-flight mass spectrometry (MALDI-TOF-MS). Homology search using MASCOT revealed high level of similarity in protein spots in both in vitro and in vivo root samples. This suggests that though in vitro roots are developed independent of shoot organs, they appear to have a similar developmental process as that of in vivo roots. This is the first report on establishment of a 2-DE reference proteome map of Withania somnifera roots and on the comparison of in vitro and in vivo root samples.

Key words: Withania somnifera, ashwagandha, in vitro roots, proteome, two dimensional electrophoresis, peptide mass fingerprinting.