Mutant analysis on the basis of phenotype and genotype.

1. Characterization of an Arabidopsis thaliana promoter trap line showing constitutive
expression of the GUS reporter gene
Sharma Pooja 1, M. Ravikumar 1, Chandra Ankita1, Gupta Navin 1, Borah Priyanka 1,
kumar Vajinder 1, Siwach Priyanka 2, Srinivasan R.1 and Bhat S. R.1
1 National Research Center on Plant Biotechnology, New Delhi-110012, India;
2 Department of Biotechnology, Ch. Devi Lal University, Sirsa-125055, Haryana, India
email: pooja0029@gmail.com
Summary: Regulation of gene expression plays a key role in determining growth and development of organisms. A
vast majority of natural variation is now believed to result from differences in gene regulation rather than due to
differences in genes per se. In order to find novel plant promoters we developed a bidirectional promoter trap vector
which carries GUS and GFP reporter genes at the left and right borders, respectively, of the T-DNA. Using this promoter
trap vector, a large number of transgenic Arabidopsis thaliana plants were generated through Agrobacterium-mediated
transformation. Screening of T1 plants for reporter gene expression identified a line FG28, which showed GUS staining
in all plant parts such as leaf, root stem, inflorescence, ovule, anther and silique. Further examination of progeny
revealed GUS expression from seedling stage to maturity.
Methodology:
Construction of Bidirectional promoter trap vector carrying GFP at RB and GUS at LB
Transformation of Arabidopsis thaliana with Bitrap vector by floral dip method
Screening of promoter trap lines of Arabidopsis thaliana in T1 generation on MS containing
kanamycin as a selection marker.
Identification of GUS expression in different tissues of T1 population by histochemical
assay.
Cloning of the T-DNA insertion flanking sequence by Thermal Asymmetric Inter-Laced (TAIL)
PCR
Southern hybridization