Systematic knockdown of thebaine 6-O-demethylase gene in Papaver somniferum L., using virus-induced gene silencing technique

Authors

1 M.Sc., Department of Agronomy and Plant Breeding, College of Agriculture, Lorestan University, Khorramabad, I.R.Iran

2 Corresponding author, Assos. Prof., Department of Agronomy and Plant Breeding, College of Agriculture, Lorestan University, Khorramabad, I.R.Iran. E-mail: ismaili.a@lu.ac.ir

3 Prof., Department of Agronomy and Plant Breeding, College of Agriculture, Lorestan University, Khorramabad, I.R.Iran

4 Assoc. Prof., Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, I.R.Iran

Abstract

   Papaver somniferum is one of the important commercial sources for several pharmaceutical
alkaloids including the narcotic analgesics codeine and morphinane and the semi-synthetic
drugs oxycodone, buprenorphine and naltrexone. One of the downstream genes in alkaloid
biosynthesis pathway in the species is thebaine-6-O demetylase (T6ODM) that inverts thebaine
and oripavine to codeinone and morphinone, respectively. In this study, a transient silencing
construct based on virus-induced gene silencing (VIGS) technique was used to knockdown the
T6ODM expression. In this regard, cDNA of T6ODM was prepared using specific primers and
the results of reaction were sequenced for confirmation. Then, a part of resulted cDNA
(silencing fragment) was selected for cloning in pTRV vector. The silencing construct was
infiltrated to plant leaves via Agrobacterium tumefaciens. The presence of cp (coat proteine)
gene of TRV vector was assayed by PCR and the positive TRV plants were screened for
analysis of gene expression by semi quantitative and real-time PCR. Results of gene expression
analysis showed that transgenic plants had a significant decrease in expression (with average
amount of 83.71%), comparing to control plants.  

Keywords

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