Abstract
The T-DNA (transferred-DNA) region of virulent Agrobacterium tumefaciens (A. tumefaciens) strain is transferred and integrated into the plant genome, and thereby the T-DNA genes are expressed in transformed plant cells. This protocol was used to analyze the transcription start sites (TSSs) of agrobacterial T-DNA genes within plant crown gall tumor. Firstly, the stems of Arabidopsis thaliana were inoculated by A. tumefaciens strain C58 and developed crown gall tumor. Subsequently, the mRNA was extracted from the crown gall tumor and then used for amplification of 5’ cDNA ends by 5’ Rapid Amplification of cDNA Ends (5’ RACE) assay. The full-length cDNAs were generated in reverse transcription reactions and used to analyze TSSs. Here, TSSs of three oncogenes, IaaH, IaaM and Ipt were analyzed as examples. This protocol also allows for identification of TSSs of the other agrobacterial T-DNA genes that expressed in plant cells.
Keywords: Agrobacterium tumefaciens, T-DNA, 5' RACE, Transcription start site
Materials and Reagents
Equipment
Procedure
Representative data
Table 2. Cis-regulatory sequence elements within the oncogene promoters
This data was published on PLOS Pathogens (Zhang et al., 2015). Positive numbers indicate the positions downstream and negative numbers the positions upstream of the TSSs (+1). TSS is underlined. Y = C/T, W = A/T, N = A/G/C/T. 1. (Klee et al., 1984); 2. (Nester et al., 1984); 3. (Goldberg et al., 1984); 4. (Heidekamp et al., 1983); 5. (de Pater et al., 1987); 6. (Lichtenstein et al., 1984)
Recipes
Acknowledgments
I would like to thank Rosalia Deeken (Department of Molecular Plant Physiology and Biophysics, University of Wuerzburg, Germany) for supervision of this work and China Scholarship Council (CSC) for my financial support. I would also like to give many thanks to Shiqiang Gao (Department of Molecular Plant Physiology and Biophysics, University of Wuerzburg, Germany) for help, suggestion and sharing reagents.
References
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