Abstarct: The UDP-glycosyltransferase74G1 enzyme that is being coded by SrUGT74G1 involves in steviol glycosides (SGs) biosynthesis in Stevia rebaudiana Bertoni. The corresponding enzyme catalyzes the synthesis of tri-glucosylated 1, 2-stevioside by glycosylating steviol biocide at C-19 position. It plays an indirect role in the production of steviol-13-O-monoglucoside and rubusoside as byproducts with the glycosylation of steviol and steviol monoside. Stevioside (5-10% of leaf dry weight) is the most abundant SGs present in Stevia rebaudiana leads to a bitter taste. In order to improve the quality of the stevia sugar flavor, the reduction of the exxpression of stevioside by srUGT74G1 gene silencing was considered. For RNAi construct preparation, primers corresponding to partial fragment of size 110 bp of this gene were designed. The forward and reverse primers were supplemented at 5'-end with the restriction site EcoRI and KpnI to generate fragments in sense orientation. Similarly, in order to generate antisense fragment, the restriction sites of XbaI and BamHI were added to forward and reverse primers at 5'-end. Total RNA was extracted from the Stevia leaf tissue and the cDNA was synthesized. Nested polymerase chain reaction was used to amplify sense and anti-sense fragments and their sequences were confirmed in pTG19-T. Both fragments were excised from pTG19-T and cloned in pHANNIBAL.