Simultaneous over-expression and silencing of some benzylisoquinoline alkaloid biosynthetic genes in opium poppy

Opium poppy (Papaver somniferum L.) remains the only commercial source for several pharmaceutical alkaloids. In present study, opium poppy plants were genetically manipulated via VIGS technique using pTRV2-BBE (to silencing of BBE1 gene), pTRV2-COM (to simultaneous silencing of T6ODM and CODM genes) and pTRV2-BBE-COM (to simultaneous silencing of T6ODM, CODM and BBE1 genes) constructs. Also, via Agrobacterium-mediated transient expression technique, poppy plants were genetically manipulated using ACS (to simultaneous silencing of T6ODM, CODM and BBE1) and ACS-4ꞌOMT2 (to simultaneous silencing of T6ODM, CODM and BBE1 and over-expression of 4ꞌOMT2) constructs. HPLC analysis showed lower sanguinarine and noscapine levels and elevated levels of morphine in plants infiltrated with pTRV2-BBE construct. In plants infiltrated with pTRV2-COM construct, reduction in the morphine and codeine content and a substantial increase in accumulation of thebaine and papaverine were observed. A substantial increase in the accumulation of thebaine and papaverine and lower levels of morphine, codeine, sanguinarine and noscapine were observed in plants infiltrated with pTRV2-BBE-COM and ACS constructs. Plants infiltrated with ACS-4ꞌOMT2 showed lower levels of codeine, sanguinarine and noscapine and elevated levels of morphine, thebaine and papaverine. Although, previous studies reported the role of our selected genes in benzylisoquinoline alkaloids (BIAs) biosynthetic pathway, but we simultaneously engineered these genes to alter the levels of specific BIAs and take advantage of all genes at the same time. For the first time, we showed different pattern in BIAs accumulation in simultaneous gene manipulation compared with single gene manipulation in previous reports, especially for higher levels of thebaine (about 1550%) and papaverine (about 155%). We also determined potential role of BBE1 gene in noscapine biosynthesis. This is the first report of simultaneous silencing and over-expression of biosynthetic genes in opium poppy and our finding could establish vast potential for metabolic engineering in opium poppy.