TITLE:
RNAi Mediated Silencing of Gene Encoding 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase (DXR) in Centella asiatica
AUTHORS:
Richa Sharma, Ratna Kalita, Basanta Kr. Borah, Mahendra K. Modi, Priyabrata Sen
KEYWORDS:
Centella, DXR, RNAi, Gene Knockdown, Secondary Metabolite, Agro Infiltration
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.11 No.11,
November
23,
2020
ABSTRACT: Centella asiatica (L.) is
one of the most valuable medicinal plants since prehistoric times. The pharmaceutical importance of this herb is due to the
accumulation of large quantities of pentacyclic triterpenoid saponins,
collectively known as centelloids synthesized by the isoprenoid biosynthesis pathway. Biosynthesis of triterpenoid in the
plants proceeds via either of the two pathways, viz. Mevalonate (MVA)
pathway (in the cytosol) or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway
(in plastid). In Centella, the pathway
leading to the accumulation of triterpenoid is still not known or elucidated.
Thus, to know whether the MVA or MEP pathway or a cross-talk between the
pathway leads to the biosynthesis of triterpenoid, silencing the key regulatory
gene using RNAi tool, of each of the pathway and then analyze a metabolite is
an efficient approach. The key regulatory enzyme of the MVA pathway i.e. 3-Hydroxy-3-methylglutaryl-coenzyme
A reductase (HMGR) has already
been successfully silenced using RNAi tool [1]. In the present study, the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) a key regulatory enzyme in MEP
pathway is silenced. The RNAi-DXR construct in pHANNIBAL vector was cloned into a binary vector pART27 and
subsequently transformed into Agrobacterium strain AGL1. The transient analysis of the RNAi-CaDXR using semi-quantitative RT-PCR confirmed the silencing of the
endogenous DXR gene in Nicotiana and
further confirmed in Centella asiatica.
The present study is the first step aimed to delineate the MEP pathway using
RNAi silencing approach to elucidate its role in the accumulation of
triterpenoid in this important medicinal plant.