Molecular analyses of Nicotiana tabacum, transformed with rolB gene of Agrobacterium rhizogenes

Abstract

Nicotiana tabacum is known to possess various pharmacological activities and is widely used to benefit humans and animals. Transgenic technologies result in genetic and epigenetic variations in plants, leading to changes in endogenous gene expression. The rolB gene belonging to Agrobacterium rhizogenes has been recognized for various valuable biological activities in transgenic plants. This study aims to expand the knowledge regarding the medicinal properties of N. tabacum having the rolB gene as a transgene, as well as to explore the epigenetic effects of the rolB gene in transgenic plants. PCR was used to validate the integration of transgene in the plant genome and real-time qPCR was used to quantify its expression level. Several in vitro and in vivo experiments were performed to evaluate the biological activities of the transgene-expressing plants. MethylFlashTM Methylated DNA Quantification Kit was used to measure the global methylation level changes of DNA, in rolB transgenics of Nicotiana tabacum. To find the molecular background of methylation variations, the expression level of epigenetic process-related genes (NtMET1, NtA31, NtROS1, and NtDRM1) in transgenic N. tabacum resources was determined by real-time PCR. Further, the interaction of the RolB protein with the proteins involved in methylation was determined by molecular docking studies. Compared to plants without transgene, a maximum of 69% increase in phenolic content, 87% increase in flavonoid content, 79% increase in reducing power, and 55% increase in radical scavenging was observed in rolB transgenics. DNA protection assay also revealed an increase in the antioxidant potential of transgenic lines by protecting DNA from damage caused by free radicals. HPLC analysis showed that transgenic plants contain more phytochemicals than wild-type. The transgenic lines exhibited a maximum of,51% increase in anti-leishmanial potential, 35% increase in butyrylcholinesterase inhibition, and 34% increase in acetylcholinesterase inhibition activities. The rolB transgenics exhibited 65%, 31%, and 36% increases in analgesic, anti-inflammatory, and anti-depressant potential, respectively in comparison to transgene-free plants. Significant hypermethylation was observed in rolB transgenics when compared to plants without transgene integration. The results of RT-PCR showed that the rolB gene triggered a substantial rise in the expression of NtMET1 and NtA31 genes while NtROS1 and NtDRM1 did not exhibit any significant vii change in expression as compared to wild-type plants. Furthermore, the docking studies illustrated that the RolB protein can interact with the proteins involved in epigenetic changes in N. tabacum. In conclusion, the current study depicted that rolB gene can improve the pharmacological potential of N. tabacum such as antioxidant, anti-leishmanial, anti-Alzheimer’s, analgesic, anti-inflammatory, and anti-depressant activities. Moreover, the obtained results showed that the rolB gene functions in plants by inducing epigenetic modifications. Our findings suggest a new insight in fundamental research on epigenetic variations in rolB transgenics and can be used in elucidating the mechanism behind the various biomedical activities of the rolB gene in plants.