RNAi Mediated Silencing of Gene Encoding 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase (DXR) in &amp;lt;i&amp;gt;Centella asiatica&amp;lt;/i&amp;gt;

Richa Sharma,Mahendra K Modi,Basanta Kr Borah,Priyabrata Sen,Ratna Kalita

doi:10.4236/ajps.2020.1111124

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.

Highlights

Centella asiatica (L.) Urban is a herbaceous medicinal plant from Apiaceae family native to Southeast Asian countries such as India, Sri Lanka, China, Indonesia, and Malaysia as well as South Africa and Madagascar [2]
RNA interference (RNAi) an efficient tool to knock down a gene [12] can be definitely applied to suppress the key regulatory gene in both MVA and methyl-D-erythritol 4-phosphate (MEP) pathway and thereby analyzing the accumulation of metabolite which shall provide us with knowledge of the pathway which is responsible for the synthesis of the key compound
Prior knowledge of the pathway which causes accumulation of the secondary metabolite in medicinal plant Centella and their regulation is an important prerequisite before any molecular intervention is done

Summary

Introduction

Centella asiatica (L.) Urban is a herbaceous medicinal plant from Apiaceae family native to Southeast Asian countries such as India, Sri Lanka, China, Indonesia, and Malaysia as well as South Africa and Madagascar [2]. In Centella, the pathway leading to the accumulation of triterpenoid is still not known so many promising tools to up-regulate the key components that couldn’t be applied to the crop. RNAi an efficient tool to knock down a gene [12] can be definitely applied to suppress the key regulatory gene in both MVA and MEP pathway and thereby analyzing the accumulation of metabolite which shall provide us with knowledge of the pathway which is responsible for the synthesis of the key compound. 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) (EC1.1.1.267), the key enzyme that regulates MEP pathway [13] [14] is being silenced. The present study is the first step aimed at knock-down DXR, a key regulatory enzyme of the MEP pathway and further confirmation through transient analysis

Methods

Results

Conclusion