Silencing of final gene involved in biosyntesis of papaverin and sanguinarin alkaloids (DBOX) using VIGS technique in Papaver somniferum L.

Aim: In this study, the effect of silence on the expression of the key gene expression of DBOX (which encodes the final enzyme for the synthesis of two alkaloids, Sanguinarin and papaverin) was used by VIGS technique in a species of poppy (Papaver somniferum L.).
Material and A fragment of 350 pairs of alkali from the DBOX gene sequence (within the range of 1112-1462bp) was selected based on the highest number of siRNA production with 21 nucleotides length. After cloning this segment into the pTZ57R/T vector and transferring the vector to pTRV2 viral vector, Agrobacterium inoculation liquid containing silencer was injected into the poppy plants leaves. Primary transgenic plants were selected by PCR reaction using a protein-binding protein coding gene primer (CP) and secondary screening was performed by semi-quantitative PCR technique. In the next step, the samples with the maximum silence (lowest expression) of the gene were examined by real-time RT-PCR technique.
Cloning accuracy in pTZ57R/T and pTRV2 plasmids were confirmed using PCR and enzymatic digestion. Based on the results of semi-quantitative PCR, 5 transgenic plants were selected with the lowest expression for DBOX gene. Based on semi-quantitative PCR results, 5 transgenic plants with the lowest expression were selected for DBOX gene. The results of real-time RT-PCR showed averagely decrease of 81% in the expression of DBOX gene transcriptions in transgenic plants compared to control plants (inoculated with the pTRV2 empty plasmid).
The results generally showed that the VIGS technique could successfully reduce the DBOX gene expression in poppy plants. In addition, the results obtained for this gene can be used to understand the biosynthetic pathway of poppy alkaloids and transgenic plants for metabolic engineering purposes.