title: Assessing the tobacco-rattle-virus-based vectors system as an efficient gene silencing technique in Datura stramonium (Solanaceae).
abstract: Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium.
authors: National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
authors: Mohammad Reza
authors: MR
authors: Eftekhariyan Ghamsari
authors: Farah
authors: F
authors: Karimi
authors: Seyed Latif
authors: SL
authors: Mousavi Gargari
authors: Seyed Ali
authors: SA
authors: Hosseini Tafreshi
authors: Seyed Alireza
authors: SA
authors: Salami
mesh_terms: D000470:Alkaloids / Q000032:analysis; D002338:Carotenoids / Q000032:analysis; D002734:Chlorophyll / Q000032:analysis; D013286:Datura stramonium / Q000235:genetics*; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D018515:Plant Leaves / Q000737:chemistry; D010942:Plant Viruses / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D000470:Alkaloids; D002734:Chlorophyll; D002338:Carotenoids; D010088:Oxidoreductases; C022398:phytoene dehydrogenase