Abstract
Stevia leaves contain various components, such as flavonoids, labdanes, chlorophylls, sterols, triterpenoids, mono-disaccharides, organic acids and inorganic salts. Stevia is known to accumulate diterpenoid steviol glycosides, which are approximately 300 times sweeter than regular sugar. Stevioside and rebaudioside A are the main diterpenic glycosides in stevia. Steviol glycosides are the secondary metabolites responsible for the sweetness of stevia. The main objectives of the present study were to determine the concentrations of diterpenic glycosides (stevioside and rebaudioside A) in three stevia varieties (Stevia rebaudiana) via the HPLC-UV technique and to amplify the UGT76G1 gene by PCR using gene-specific primers. The expression levels of the UGT76G1 gene were determined in the three stevia varieties. The PCR products were sequenced and analyzed, and the nucleotide sequences of the UGT76G1 gene were submitted to GenBank and assigned to the following three varieties: Egy1 (MH087463), China1 (MH087464) and Sponti (MH087465). Cluster analysis was used to separate the three varieties into two major clusters based on their phylogenetic relationship. In addition, chemical analysis was carried out to evaluate stevioside and rebaudioside A. The present study concluded that Egy1 and Sponti are closely related varieties as they fall in the same cluster, while China1 forms a separate cluster. Bioprospecting studies could be useful for selection of superior ecotypes of Stevia rebaudiana.
Highlights
Stevia plants are an important source of commercial steviol glycosides (SGs)[1]
The addition of the C-13-glucose to steviol is catalyzed by UGT85C2, first yielding steviolmonoside and steviolbioside; the addition of the C-19-glucose is catalyzed by UGT74G1, yielding stevioside[30,31]; and glucosylation of the C-3′ of the glucose at the C-13 position is catalyzed by UGT76G1, yielding rebaudioside A27,32
The results shown in Table (2) and Fig. (1A–C) indicate that the highest stevioside content was observed in the Sponti variety (21.46%), followed by China[1] (0.18%) and Egy[1] (12.27%)
Summary
Stevia plants are an important source of commercial steviol glycosides (SGs)[1]. Eight different steviol glycosides are produced in stevia plants. Plant UDP-glycosyltransferases (UGTs) are a unique group of enzymes that transfer sugar residue from an activated donor to an acceptor molecule[14,15,16,17,18,19]. In Stevia, UDP-glycosyltransferases are involved in the production of steviol glycosides, compounds that are unique in the plant world due to their intense sweetness and high concentration in leaves[25,26]. To differentiate between three stevia varieties, biochemical analysis of the three varieties was carried out, to determine the concentrations of both stevioside and rebaudioside A in stevia leaves using HPLC. The UGT76G1 gene was characterized via gene sequencing to determine the genetic similarity of the three stevia varieties, and the obtained sequences were submitted to GenBank to identify accession numbers
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