Cloning, Characterization and RNAi mediated silencing of gene encoding 1-deoxy-D-xylulose 5-phosphatereductoisome rase (DXR) in Centella asiatica
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Date
2019-01
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AAU, Jorhat
Abstract
Centella asiatica (L.) is one of the most valuable medicinal plants which
belong to the family Apiaceae. The medicinal importance of this green leafy
vegetable is known 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-Derythritol
4-phosphate (MEP) pathway (in plastid). In Centella, the pathway leading
to the accumulation of triterpenoid is still not known. Thus, to know whether the
MVA or MEP pathway or contribution of both has a role in the biosynthesis of
triterpenoid, silencing the key regulatory enzyme gene using RNAi tool, of each of
the pathway and then to analyze a metabolite is an efficient approach. In our lab,
HMGR (a key enzyme of MVA pathway) RNAi construct has already been designed,
confirmed by RT-PCR and validated by Agro-infiltration. 1-deoxy-D-xylulose-5-
phosphate reductoisomerase (DXR) play a role in catalyzing the first committed step
of the MEP pathway. The present study is the first step aimed to delineate the MEP
pathway using RNAi silencing approach to knock down rate limiting 1-deoxy-Dxylulose-
5-phosphate reductoisomerase (DXR) enzyme. The full-length DXR gene
sequence (JQ965955) of Centella has been characterized using in silico approach.
CaDXR is a 1425bp ORF encoding a peptide of 474 amino acids and of molecular
weight of 51.5 KDa. Multiple sequence comparison using MEGA tool showed the
presence of two NADPH binding motif, two substrates binding motif, and one
cleavage site motif. In this study, the 3-D structure of CaDXR was identified and
validated along with this molecular dynamics simulation and finally docking with
cofactor NADPH was done. The expression analysis suggests that CaDXR is
differentially expressed in different tissues (with maximal expression in node and
lowest in the roots). Our result suggests that nodes may be crucial to terpenoid
biosynthesis in Centella asiatica. The RNAi-DXR construct was designed using the
pHANNIBAL vector and subsequently cloned into a binary vector pART27. The
binary vector pART27 containing RNAi-CaDXR construct was transformed into
Agrobacterium strain AGL1. The transient analysis of the RNAi-CaDXR using semiquantitative
RT-PCR confirmed the silencing of the endogenous DXR gene in
Nicotiana and further confirmed in Centella asiatica. Thus, further incorporation of
both the RNAi construct (HMGR and DXR) in transformed Centella shall shed light
into the pathway that leads to the synthesis of principal secondary metabolites i.e
centelloids.