Cloning of RNAi BBE Gene Fragments into the pGSA1285 Silencing Vector in Poppy Plant

Poppy is one of the most important herbs in the pharmaceutical industry. It produces a wide range of isoquinoline alkaloids. Genetic manipulation of important enzymes involved in metabolic pathways could change poppy alkaloids. One of the techniques used for plant manipulation is gene silencing using RNA interference. In this study, RNAi technique was used to investigate the effect of gene silencing of Berberine Bridge Enzyme (BBE) gene on the major profile of Morphinan alkaloids network. After identification and isolation of BBE gene using specific primers, specific region of gene was selected for silencing. The target gene was cloned in T / A vector and sequenced. The fragment was then separated from the T / A vector by digestion. The silencing fragment was cloned in pGSA1285 vector in the sense strand. After confirmation of the presence of silencing fragment in the sense strand, pGSA1285 vector was digested again with digestive enzymes and the silencing fragment was cloned in antisense strand. Then, the whole silencing construct containing sense intron and antisense was confirmed by digestion.