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Analysing virulence gene expression profile of Agrobacterium Tumefaciens under different external factor through qPCR

Student name: Ms Priya Vaish
Guide: Dr Shashi Bhushan Tripathi
Year of completion: 2018
Host Organisation: School of Biotechnology, Jawaharlal Nehru University, New Delhi
Supervisor (Host Organisation): Dr Manoj K. Sharma
Abstract: Agrobacterium a gram-negative soil a bacterium that belongs to phylum Proteobacteria. Crown Gall disease is characterised by tumorous phenotype which is caused by Agrobacterium in dicotyledonous plants. Agrobacterium causes tumor by the following process: Segment of Ti-plasmid DNA (T-DNA/transferred DNA) from Agrobacterium gets transferred into the host genome which genetically transforms the host. Introduction of foreign genes into the plant cell and the regeneration of transgenic plants is possible mainly due to this property of Agrobacterium Tumefaciens is used extensively for the. Agrobacterium’s Ti-plasmid have Virulence (vir) region that plays most important role during transformation. At least these six (VirA, VirB, VirC VirD, VirE and VirG) essential operons and these two (VirF and VirH) less essential operons are present in this vir regions, and encoding approx. 30 proteins and those proteins required plant signalling molecules for the activation of this system.

Objective of this study is to analyse the expression pattern of vir genes of agrobacterium under different external factor and those factors can be different types of plants signalling molecules. Based upon the literature survey we have selected 9 candidate vir genes (VirA, VirG, VirB1, VirB4, VirD2, VirE2, VirC1, VirB7, VirD1) from 30 vir genes. qPCR primers that were Gene-specific were synthesized. These primers was then used to check their expression in external factor which we have selected from literature survey, for gene expression study we have used the Quantitative Real-time PCR (qPCR) which is one of the robust technique, in which PCR product can be measured in real-time.

Keywords: qPCR, Vir genes, Transformation, Plant Signalling molecules