Gene cloning and functional analysis of an iridoid oxidase gene in Rehmannia glutinosa
Rehmannia glutinosa is one of the commonly used Chinese herbal medicines,which has activities of heat-clearing,blood-cooling,Yin-nourishing,and body fluid-promoting.Iridoid glycosides are the main bioactive in R.glutinosa.Iridoid oxidase is a key rate-limiting enzyme in the biosynthetic pathway of iridoid glycosides.In this study,an iridoid oxidase gene RgIO was screened based on the transcriptome data,followed by bioinformatics analysis,expression characteristic detection,and subcellular localization analysis.The results show that the coding region of RgIO is 1 536 bp,with 511 amino acids encoded,and the molecular weight is about 58 258.01.The protein sequence of RgIO contains the conserved domains and motifs of cytochrome P450 oxidases.RgIO has the highest sequence identities with its ortholog proteins in Striga asiatica,Striga hermonthica,and Centranthera grandiflora and has good sequence identities(77.28% )with Catharanthus roseus CrIO.RgIO shows specific expression in the leaf of R.glutinosa.In response to MeJA induction,the expression of MeJA in leaves and roots after treatment increases by 3.15 and 1.3 times at 3 h and 6 h,respectively.The result of subcellular localization shows that RgIO is distributed in the endoplasmic reticulum.Agrobacterium-mediated transient expression of RgIO gene in leaves of R.glutinosa makes the content of catalpol increase by 0.82 times compared with the transient expression of the empty vector.This study provides a key target gene for the molecular regulation and biosynthesis of catalpol in R.glutinosa and lays a foundation for revealing the complete biosynthetic pathway of catalpol.
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