Co-silencing of Thebaine 6-O-demethylase and Codeine O-demethylase in Papaver somniferum L., Using Virus-Induced Gene Silencing

Document Type : research

Authors

1 MSc Student, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad

2 Associate Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad

3 Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad

Abstract

Papaver somniferum remains the sole commercial source for narcotic and variety of semisynthetic drugs. Thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM) genes are related the dioxygenase family that occur at the end of biosynthesis of these alkaloids in this plant. For silencing of CODM and T6ODM genes in Papaver somniferum, a TRV2-based construct containing a conserved sequence of DIOX from the coding region of T6ODM and CODM was designed for simultaneously silencing of two mentioned genes. Emerging first leaves of 2 to 3-week-old seedling were infiltrated with Agrobacterium tumefaciens harboring silencing construct, or the empty vector (TRV2) as a control. Analysis of gene expression at the transcript level by real-time quantitative PCR (polymerase chain reaction) technique showed 86 and 87 percent reduction in gene expression of CODM and T6ODM compared with control plants, respectively. This study demonstrated the efficiency of this method for knockdown the transcript levels of specific BIA biosynthetic genes.

Keywords

References

Allen, R. S., Miller, J. A., Chitty, J. A., Fist, A. J., Gerlach, W. L. and Larkin, P. J. 2008. Metabolic engineering of morphinan alkaloids by over‐expression and RNAi suppression of salutaridinol 7‐O‐acetyltransferase in opium poppy. Plant Biotechnology Journal, 6: 22-30.
Burch‐Smith, T. M., Anderson, J. C., Martin, G. B. and Dinesh-Kumar, S. P. 2004. Applications and advantages of virus‐induced gene silencing for gene function studies in plants. The Plant Journal, 39(5): 734-746.
Dang, T. T. and Facchini, P. J. 2012. Characterization of three O-methyltransferases involved in noscapine biosynthesis in opium poppy. Plant Physiology, 159(2): 618-631.
Dinesh-Kumar, S., Anandalakshmi, R., Marathe, R., Schiff, M. and Liu, Y. 2003. Virus-induced gene silencing. In Plant Functional Genomics, pp. 287-293. Springer.
Facchini, P. J. and Park, S. U. 2003. Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry, 64(1): 177-186.
Frick, S., Chitty, J. A., Kramell, R., Schmidt, J., Allen, R. S., Larkin, P. J. and Kutchan, T. M. 2004. Transformation of opium poppy (Papaver somniferum L.) with antisense berberine bridge enzyme gene (anti-bbe) via somatic embryogenesis results in an altered ratio of alkaloids in latex but not in roots. Transgenic Research, 13: 607-613.
Frick, S., Kramell, R. and Kutchan, T. M. 2007. Metabolic engineering with a morphine biosynthetic P450 in opium poppy surpasses breeding. Metabolic Engineering, 9: 169-176.
Hagel, J. M. and Facchini, P. J. 2010. Dioxygenases catalyze the O-demethylation steps of morphine biosynthesis in opium poppy. Nature Chemical Biology, 6(4): 273-275.
Hagel, J. M. and Facchini, P. J. 2013. Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant and Cell Physiology, 54(5): 647-672.      
Hileman, L. C., Drea, S., Martino, G. D., Litt, A. and Irish, V. F. 2005. Virus‐induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy). The Plant Journal, 44(2): 334-341.
Inui, T., Tamura, K. I., Fujii, N., Morishige, T. and Sato, F. 2007. Overexpression of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in benzylisoquinoline alkaloid biosynthesis in cultured Eschscholzia californica. Plant and Cell Physiology, 48: 252-262.
Kempe, K., Higashi, Y., Frick, S., Sabarna, K. and Kutchan, T. M. 2009. RNA suppression of the morphine biosynthetic gene salAT and evidence of association of pathway enzymes. Phytochemistry, 70: 579-589.
Larkin, P. J., Miller, J. A., Allen, R. S., Chitty, J. A., Gerlach, W. L., Frick, S., Kutchan, T. M. and Fist, A. J. 2007. Increasing morphinan alkaloid production by over‐expressing codeinone reductase in transgenic Papaver somniferum. Plant Biotechnology Journal, 5: 26-37.
Liu, Y. 2002. Virus‐induced gene silencing in tomato. The Plant Journal, 31(6): 777-786.
Millgate, A. G., Pogson, B. J., Wilson, I. W., Kutchan, T. M., Zenk, M. H., Gerlach, W. L., Fist, A. J. and Larkin, P. J. 2004. Analgesia: morphine-pathway block in top1 poppies. Nature, 431: 413-414.
Ounaroon, A., Decker, G., Schmidt, J., Lottspeich, F. and Kutchan, T. M. 2003. (R, S)‐Reticuline 7‐O‐methyltransferase and (R, S)‐norcoclaurine 6‐O‐methyltransferase of Papaver somniferum-cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. The Plant Journal, 36(6): 808-819.
Ratcliff, F. 2001. Technical advance: tobacco rattle virus as a vector for analysis of gene function by silencing. The Plant Journal, 25(2): 237-245.
Robertson, D. 2004. VIGS vectors for gene silencing: many targets, many tools. Annual Review Plant Biology, 55: 495-519.
Samanani, N., Liscombe, D. K. and Facchini, P. J. 2004. Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. The Plant Journal, 40(2): 302-313.
Wijekoon, C. P. and Facchini, P. J. 2012. Systematic knockdown of morphine pathway enzymes in opium poppy using virus‐induced gene silencing. The Plant Journal, 69(6): 1052-1063.
Ziegler, J. 2005. Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis. Planta, 222(3): 458-471.
Volume 8, Issue 1
June 2017
Pages 1-10

Files

Share

How to cite

Statistics